Text

PRM-031-004 - 53FR02853 - Gene-Trak Systems: Filing of Petition for Rulemaking
	ML23153A044
Person / Time
Issue date:	02/02/1988
From:	; NRC/SECY
To:	;
References
	PRM-031-004, 53FR02853
	Download: ML23153A044 (1)
	v • d • e

DOCUMENT DATE:

TITLE:

CASE

REFERENCE:

KEYWORD:

ADAMS Template: SECY-067 02/02/1988 PRM-031-004-53FR02853 - GENE-TRAK SYSTEMS: FILING OF PETITION FOR RULEMAKING PRM-031-004 53FR02853 RULEMAKING COMMENTS Document Sensitivity: Non-sensitive - SUNS! R~view Complete

PAGE 1 OF 2 STATUS OF RULEMAKING RECORD 1 OF 1

PROPOSED RULE:

PRM-031-004 RULE NAME:

GENE-TRAK SYSTEMS: FILING OF PETITION FOR RULEMAKING PROPOSED RULE FED REG CITE:

53FR02853 PROPOSED RULE PUBLICATION DATE:

02/02/88 NUMBER OF COMMENTS:

6 ORIGINAL DATE FOR COMMENTS: 04/04/88 EXTENSION DATE:

I I

FINAL RULE FED. REG. CITE: 54FR10550 FINAL RULE PUBLICATION DATE: 03/14/89 NOTES ON: PETITION CONCERNED USAGE OF PHOSPHORUS-32 IN FOOD LABORATORIES. PE ATUS

TITION WAS WITHDRAWN BY GENE-TRAK. SECRETARY SIGNED WITHDRAWAL NOT F RULE: ICE ON 3/9/89.

FILE LOCATED ON P-1.

PRESS PAGE DOWN OR ENTER TO SEE RULE HISTORY OR STAFF CONTACT PRESS ESC TO SEE ADDITIONAL RULES, (E) TO EDIT OR (S) TO STOP DISPLAY PAGE 2 OF 2 HISTORY OF THE RULE PART AFFECTED: PRM-031-004 RULE TITI,,E:

-ROPOSED RULE*

GENE-TRAK SYSTEMS: FILING OF PETITION FOR RULEMAKING PROPOSED RULE DATE PROPOSED RULE

DOCKET NO. PRM-O31-OO4 (53FRO2853)

DATE DOCKETED In the Matter of GENE-TRAK SYSTEMS: FILING OF PETITION FOR RULEMAKI NG DATE OF TITLE OR DOCUMENT DESCRIPTION OF DOCUMENT

03/14/88 03/10/88 COMMENT OF DEPARTMENT OF THE AIR FORCE

{LAWRENCE DONOVAN) {

2) 11/25/87 11/18/87 PETITION FOR RULEMAKING FROM GENE-TRAK SYSTEMS 03/24/88 03/21/88 COMMENT OF ILLINOIS DEPARTMENT OF NUCLEAR SAFETY

{TERRY R. LASH) {

3) 01/28/88 01/27/88 FEDERAL REGISTER NOTICE:

FILING OF PETITION FOR RULEMAKING 03/31/88 03/08/88 COMMENT OF COLORADO DEPARTMENT OF HEALTH

{ALBERT J. HAZLE) {

4) 02/24/88 02/20/88 COMMENT OF MARVIN I. LEWIS {

1) 03/31/88 03/18/88 COMMENT OF FL. DEPT. OF HEALTH/REHABILITATIVE SERV.

(J. DANIEL NASH) (

5) 03/14/_88 03/10/88 COMMENT OF DEPARTMENT OF THE AIR FORCE (LAWRENCE DONOVAN, MAJOR, USAF) (

2) 05/02/88 04/27/88 COMMENT OF DIVISION OF RADIOLOGICAL HEALTH (HAROLD R. BORCHERT, DIRECTOR) (

6) 03/24/88 03/21/88 COMMENT OF ILLINOIS DEPARTMENT OF NUCLEAR SAFETY (TERRY R. LASH) (

3) 03/25/88 03/18/88 COMMENT OF STATE OF FLORIDA (J. DANIEL NASH) (

5) 11/25/87 11/18/87 PETITION FOR RULEMAKING - GENE-TRAK SYSTEMS (VOLTZ-DOW) 01/28/88 01/27/88 FEDERAL REGISTER NOTICE - RECEIPT OF PETITION FOR RULEMAKING 03/08/89 02/28/89 GENE-TRAK SYSTEMS WITHDRAWS PETITION FOR A RULE CHANGE

DOCKET NO. PRM-031-004 (53FR02853)

DATE DATE OF TITLE OR DOCKETED DOCUMENT DESCRIPTION OF DOCUMENT 03/09/89 03/09/89 NRC SUBMISSION TO THE FEDERAL REGISTER FOR WITHDRAWAL OF PETITION 03/31/88 03/08/88 COMMENT OF STATE OF COLORADO (ALBERT J. HAZLE) (

4) 05/02/88 04/27/88 COMMENT OF STATE OF NEBRASKA (HAROLD R. BORCHERT, DIRECTOR) (

6) 02/24/88 02/20/88 COMMENT OF MARVIN I. LEWIS (

1) 03/08/89 02/28/89 WITHDRAWAL OF PETITION BY GEORGE PARSONS, PH.D.

GENE-TRAK SYSTEMS DUE TO INTRODUCTION OF NEW PRODUCTS AND MARKETING STRATEGY 03/09/89 03/09/89 WITHDRAWAL OF PETITION FOR RULEMAKING

[7590-01]

a9 MAR -9 P 1 :45 NUCLEAR REGULATORY eci0iMMISSION lJ CFiT I

f 10 CFR Part 31 fl.:. t..

[Docket No. PRM-31-4 ]

GENE-TRAK Systems; Withdrawal of Petition For Rulemaking AGENCY:

Nuclear Regulatory Commission.

ACTION:

Petition for rulemaking; withdrawal.

SUMMARY

The Nuclear Regulatory Commission (NRC) is withdrawing, at the petitioner's request, a petition for rulemaking filed by GENE-TRAK Systems (PRM-31-4).

The petition for rulemaking requested that the NRC establish that 100 microcuries of phosphor.us-32 used in GENE-TRAK Sal monella and Listeria assays by a food laboratory is an exempt quantity under a general license according to 10 CFR 31.11.

The petitioner is withdrawing the petition because of the introduction of new products and resulting changes in marketing strategy.

ADDRESSES:

A copy of the petitioner's letter requesting withdrawal of the petition is available for public inspection or copying for a fee in the NRC Public Document Room, 2120 L Street, NW., lower level of the Gelman Building, Washington, DC 20555.

A single copy of the petitioner's letter requesting the withdrawal of

J I. S. NUCLEAR RFGULATO Y COMMI SSION DOCKE.,..ING & SERVICE SECTION O"FICF. OF THE SE::RETARY OF THE CO, *\\MISSION DoctJme nl Statistics Postmark Date Copies C:.ecei ve1

/ -

Add' I Cc,i s Reproduced 3 -------

Special Distribution

/l-Vl- ?JDS p r

2 petition may be obtained by writing the Regulatory Publications Branch, Division of Freedom of Information and Publications Services, Office of Administration, U.S. Nuclear Regulatory Commission, Washington, DC 20555.

FOR FURTHER I NFORMATION CONTACT:

Michael T. Lesar, Acting Chief, Rules Review Section, Regulatory Publications Branch, Division of Freedom of Information and Publications Services, Office of Administration, U.S. Nuclear Regulatory Commission, Washington, DC 20555, Telephone:

301-492-7758 or Toll Free:

800-368-5642.

Dated at Rockville, Maryland, this q th day of March 1989.

or the Nuclear Regulatory Commission.

on.

31 New York Avenue

=.=GENE-TRAK

~SYSTEMS Framingham, Massachusetts 01701 508-872-3113 D'JCKETEO USNRC Fax: 508-879-6462 REGULATORY PUB' :c.ATIOMS eP.

~ I* i ii ht**

'89 MP.R -3 P2 :16

'89.MAR -8 P 5 :46 February 28, 1989 Director Division of Rules and Records U.S. Nuclear Regulatory Commission Hashington, D. C.

20555

Dear Sir:

r,F~,

ooc'KL I l'J l *'

t;t~. Nv-*

v. f Due to introduction of new products and resultant changes in our marketing strategy, we would like to withdraw our petition for a rule change. This rule change was requested in a letter to you dated November 18, 1988 from Ms. Karenlee Dow and has been designated as #PRM-31-4.

Thank you for your help. Please feel free to call me if you have any questions.

Sincerely,

,ffi~11~~

/ George Parsons, Ph.D.

Radiation Safety Officer cc:

Mr. Michael Lessar (Office of Admi nistration)

Mr. Harvey Scott (Office of Research)

GP:gmc/100lk

(Alt 1W1ULA foriv ~6MM(SSta&

DOCKETING & SERVICE SECTION OFFICE OF THE SECRET ARY.

OF THE COMMISSION Document Statistics Poshnark Date JvjJ

_ __..:~------

1:o pies Received ---=---------

Ii'. d d' I Copies Reproduced ------

IRecial Distribution

STATE OF w

NEBRASKA DEPARTMENT OF HEALTH DOtKETF*

U-St HC KAY A. ORR GOVERNOR DOCKE GREGG F. WRIGHT, M.D., M.Eo.

T NUMBER DIRECTOR PETITION RULE PRM ~I _ ~ "88 HAY -2 p 5 :50

( S':1 F~ )..°15 ~)

April 27, 1988 Secretary of the Commission U.S. Nuclear Regulatory Commission Washington, D.C. 20555 Attention:

Docketing and Service Branch Re:

Notice of receipt of petition for rulemaking from GENE-TRAK Systems.

Docket No. PRM-31-4.

1)

The general license for use of radioactive material for certain in-vitro clinical or laboratory testing is restricted to a physician, veterinarian in the practice of veterinary medicine, clinical labora-tory or hospital in the state of Nebraska.

The Division of Radio-logical Health would not recognize an in-vitro general license for use in the food industry and it's regulators.

The in-vitro general license is acceptable in the medical environment, because of the highly trained technical personnel as well as numerous regulatory review processes.

2)

A specific license is a mechanism utilized by licensing agencies to require accountability of companies using radioactive materials.

Procedures for receipt, use, handling, disposal, and routine surveys must be submitted prior to first shipment of radioactive materials.

A general license would relieve the licensee from submitting these procedures.

This loss of accountability could have health and safety implications.

3)

Issuance of a general license bypasses the regulatory review of train-ing and experience for users of the Phosphorous-32 salmonella kit.

Training is a key consideration for safe handling of radioactive materials.

Health and safety of personnel could be compromised if the issuance of a general license bypasses review of a user's training.

Safety is promoted by adequate training programs.

The Division of Radiological Health, Nebraska Department of Health requires 30-40 hours of formal training in radiation safety for all users of small quantities of radioactive materials.

(continued) 301 CENTENNIAL MALL SOUTH P. 0. BOX 95007, LINCOLN, NEBRASKA 68509-5007 AN EQUAL OPPORTUNITY/ AFFIRMATIVE ACT

~

M"d~cfll4'.,by card.* P..:~z:-.&

U. $. NUCtEAR R'CI" ~ TORY COMMJSSlot.

DOCKf't ING & $ER'i""E SECT ION CFr iCt OF TH> ' f~ l?fTA.RY 0~ iHE CCM1'\\l '.:.S!ON Postm5rk Date

'-( - 2. "'1 ":" fE' Copies Rcc-0* ve*.1 f

Add ' I Co p1,

...: ,:-,-L:ccd l-.

Secretary of the Commission Continued ****

Page 2 April 27, 1988 Thank you for the opportunity to provide comments on this issue.

Sincerely,

~

?;-.:::.,..,~

Harold R. Borcher, Director Division of Radiological Health Nebraska Department of Health HRB:efn

DOCKET NUMBER PETITION RULE PRM -.3l--4{Z) -t(i:,<*/l"--

{ff.3 ~

,,,t?'.!F-3

. -~

f,I STATE OF FLORIDA 00u~*l'JR 1[-D -n t DEPARTMENT OF HEALTH AND REHABILITATIVE SERVICES OFFICE O F RADIATION CONTROL

aa HAR 31 p 4 :18 RADIOACTIVE MATERIALS PROGRAM Donald A. Nussbaumer, Assistant Director for State Agreements Program State, Local and Indian Tribe Program Mail Stop 3-D-23 U.S. Nuclear Regulatory Commission Washington, DC 20555

Dear Mr. Nussbaumer:

March 18, 1988 OFFICE o: Sc. Lf:.:it.h ~

OOCKfi 1NG t, SE fl' f': f.

BR.llN(: ~

DOCKETED USNRC RULES & PROCEDURES BR nRR:J\\OH

'88 HAR 25 P2 :15 This letter addresses our comments on the petition to add 100 microcuries of phosphorus-32 (P-32) to the In-Vitro general license specified in 10 CFR 31.11.

Without more elaborate dose estimates we do not feel that P-32 should be added to the general license described in 10 CFR 31.11 for the following reasons.

1.

The maximum range of the 1.71 MeV beta is 790.15 mg/ cm2 which is approximately 20 feet in air and 0.31 inches in water. (Calculations attached)

This implies that special shielding is required.

2.

The maximum permis s ible body burden is only 30 microcuries.

In addition, the maximum permissible1burden for the bone critical 0rgan is only 6 microcuries

3.

An information sheet from a major di s tributor (enclosed) indicates very high dose rates from one millicurie of P-32.

They also indicated that one should wear personnel monitoring when using P-32, preferably ring badges.

In addition, they indicate that while 60% of P-32 ingested is excreted within 24 hours2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months , only 1% per day is excreted after the second or third day.

1317 W INEWOOD BLV D.

T ALLAHASSEE, FL 32399-0700 * (904) 487-2437 BOB MARTINEZ, GOVERNOR

'-I I I

9;, 'NUCLEAR ~E(';f.lL.ATORY COt'.\\NdSSIO~

D0Cfl'Tli I(';

SE?VICE SECTION

(,;fi:C.,..,; iHf ! f,1<::~.o\\RY r.__,y,:'i:,., :, ".~ f*Jt,1 osflflU~ 0~, *

!opie~ Rr:, ** *,.

'Add' I (-.~.. **.

..***:*J cs

z._---

.§,7 iJ:r*',,

/J)_J.

S -~

Donald A. Nussbaumer March 18,1988 Page Two The general license 10 CFR 31.11 allows the use of radioactive material without personnel monitori ng.

Since no other dose assessments have been presented, we do not feel that justification has been shown to include P-32 as part of the general license.

If you have any questions, please contact us.

Sincerely,

{2-4~

J. Daniel Nash Public Health Physicist Manager MNS/jo Enclosures

1. Radiological Health Handbook, Revised Edition January 1970, page 206.

Calculations2 Physical Data:

P-32 E (beta}= 1.71 Mev 3

Densit~axair:

D.

= 1.293 mg/ cm 3 Density water: Daitr

= 1000 mg/ cm wa er Formulas:

l. R = 412[E<l.265 - 0.0954ln(E)>]

R = Range in units of mg/ cm2 E = Maximum Beta Energy in units of MeV 2

3

2. R d'

= R(mg/ cm )/D d' (mg/ cm )

me 1um me 1um t

R d'

= range in centimeters for that medium D me 1um D

f d'

th b

1*

medium=

ens1ty o me 1um e

eta 1s trave 1ng R = 412[ 1. 71 <1.265 - 0.0954ln(l.71}>]

412[1.11< 1 -214 >]

412[1.918~

790 mg/ cm R air= 790/ 1.293 = 611 cm (20 feet)

I R water= 790/ 1000 = 0.79 cm (0.31 inches}

through.

2. Radiological Health Handbook, Revised Edition January 1970, pages 29,66,239.

_Phosphorus-32: Handling and Hazart!S 0

p 32 "

The-marked increasr in the* U!-' of Phosrtioru!- 3?

32p I...~ I ** J, and the* rrlatively larE<* sh1pmC'nts havE' prompted the*

.... I l 11#,. I:,." *

.,. l*e

.....,. ~.*,.,.,.....

lo following rc*mindc-r concerninr the* hazard~ a~so c1ated v.-1t h 1111s hard beta em11tN.

~. ~-

~-

~

'r

~...

~

Maximum Enercy: 1.71 MeV (beta particle)

~

f"*....,...

,:-* **~

t,;. ~*. !--:-

!of:

!,<(

~)'

~;-:-

I,()<-

l> *-*

".,. 4~1"1

3'-

,:iF- **~

OI-,..,,..

Maximum *Range in Air: Approximately 18 feet

?'

)I,

)'(

,,., ~-

)U 3:,,

l.

t ;,,

31; 3C" -

w.

11,.:.,....,,...

Maximum Range in Water: Approximately 0.3 inches

. ~

~, :;,

1*~

1*0 134 1:¥...

C 3;

'111 10* ~; 1r 19, la!,

1~'

17$

lt 17*

170 11,t 16:

15,11 1!,~

1~*

Uf Critical Organ: Soluble forms -

Bone

r 1..

,40 137 1)"

,x, 1;,e,,.,,,

Insoluble forms -

Lung. G.I.

11) 11(1 IOf 10-,

10, 11)'

-~

09~

Ot'.-

D9J D!>'.

Of*

Of" (II,<

Of

~.

011*

07t 077 07~

07:,

c,*,

117{

01,*

DOSE RATES BODY DOSE Dose rates from l mC1 Phosphorus-32 over l~m2 of The maximum permissible body burden of Phos-phorus-32 is 301,Ci. However, the maximum permissible burden 1or the bone critical organ is 61,Ci. Although about 60% of Phosphorus-32 tha1 is ingested is excreted within the first 24 hours2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months . only about l % per day is excreted after the second or third day following ingestion. Therefore. regular skin are:

At surface of the skin At 1cm At 10cm 2.000 rads 'hr 200 rads hr 22 rads :hr Dose rate for 1 mCi Phosphorus-32 in 1 ml of water uri ne samples should be submitted, followed by rapid analysis Dose evaluations also require knowledge of the approximate date and time the isotope was handled.

At surface 780 rads 'hr

PRECAUTIONS----------

Employ both low and high density shielding.

Avoid direct contact with skin. Use gloves.

Protect eyes from chemical splash and Film badges should be used by all personnel working with Phosphorus-32. If mill1curie quantit ies are manipulated. wrist and finger badges should also be used.

unnecessary radiation.

Use remote handling tools.

e Prevent ingestion.

Wrist badge readings serve ONLY as an indication of exposure problems,n the laboratory Relatively /ow vmst badge readings may tail to reveal high exposure to the fingers.

Isolate waste.

Europe One of the greatest hazards associated with beta emitters of this energy exists in handling uncovered vessels containing the material. For example, the surface dose rate of 1 mCi of Phosphorus-32 in 1 ml of solution is app;oximately 13 rads/minute.

This dose rate will not be appreciably reduced by attenuation in a few

centimeters of air, nor will there be much reduction by inverse square law trom a source of this kind. It is obvious that a hand or face over such an open container may receive a considerable dose of radiation in a short period of time. e New England Nuclear 5~:- A*t.c:'"*.* St*e,;: 6::,s*:-- ~*~ssa:~..:sE~'.s o::,,e Canada NEN Ct1em1:a1s G-.= -

CALL TOLL*FREE 800*22~-, 572 Tele>>. 94-0996

1...,.~assa: "1.ise::s a,..:,... :f*::a:.:,na' ~ 7-~52*9::*?51

. ~.. - -.. *-..

COLORADO DEPARTMENT OF HEALTH

~

STATE OF COLORADO 4210 East 11th Avenue DOCl<ET NUMBER Denver, Colorado 80220 PR

/ VAi Phone (303) 320-8333 PETITION RULE M-.3 -4b' March 8, 1988 Donald A. Nussbaumer Assistant Director for State Agreements Program (53 F,e, 7.¥§~

State, Local and Indian Tribe Programs Mail Stop 3-D-23 U. S. Nuclear Regulatory Commission Washington, DC 20555

Dear Mr. Nussbaumer:

DOCKETED tA~R3 119Sa DOCK£T1NG &

\\

SEffllC&1111-N SECT-r-

Roy Romer Governo r The following are the Colorado comments regarding the Federal Register notice on a petiticn for an In-Vitro general license.

(See your letter dated February 11, 1988.)

While we do not have reason to believe that the proposal clearly is inconsistent with the general purpose, intent and technical aspects of such general licenses already in NRC and Agreement State regulations, we do have a concern.

This concern is based on growing information data base that the concept of general licensees as practiced by the NRC and the Agreement States has not provided adequate control of the licensees and the materials consistent with public trust and radiation safety.

We now believe that general licenses should be deleted from the regulations and either the situation exempt from regulatory control or under a specific license.

The general license has outlived its usefulness.

e, Director Radiation Control Division AJH/cf APR l 1988

~knowledgc-d by card.,*rr.-u""I*/~-.,,-;;:, __

I

0. s. NUCLEAR r. 1 G'. 11 ;.. T (' ~ - ~OMMI SSIOH OOC~ET1r~:G " r.~?\\':c

cTtON Oir'. '

,-

C

~

Postma,~ D)*

Copies i

,.*~ :-,ARY

., ', <; <; ION

DOCKET NUMBER

. ETITION RULE PRMJ/-<f (5) 53F£2?5"J}

OOlKE,EC*

us~mc STATE OF ILLIN0IS "88 HAR 24 PS :47 DEPARTMENT OF NUCLEAR SAFETYFF1c:: vi-

~ ~*~ *.r

.:_1;,

TERRY R. LASH DIRECTOR Secretary of the Commission 1035 OUTER PARK DRIVE OOCK[T~~iN*c~( r<'v ICf.

SPRINGFIELD 62704 (217) 785-9900 March 21, 1988 U.S. Nuclear Regulatory Conunission Washington, D.C. 20555 Attention: Docketing and Service Branch Re:

Petition for Rulemaking, "In-Vitro General License," PRM-31-4 10 CFR Part 31.11 (53 Fed. Reg. 2853; February 2, 1988)

Dear Sir:

The Illinois Department of Nuclear Safety submits herewith its comments on the above-identified petition for rulemaking. The petition asks that phosphorous-32 be included in the in-vitro general license granted under 10 CFR 31.ll(a). The Department is generally in favor of granting a general l icense for such tests.

The Salmonella and Listeria detection tests discussed in the petition appear to be a major advancement for safety in the food industry. These research advances should be encouraged by safety regulatory agencies whenever possible. Establishment of a general license for use of phosphorous-32 in these tests would encourage further research by making similar products and applications economically desirable.

The Department has two concerns about the petition as presented. First, the petitioner should be aware that the inclusion of 100 µCi of phosphorous-32 in 10 CFR 31.ll(a) would not redefine this amount as an exempt quantity. Neither would an applicant for such general license be exempt from licensure, although the general license would represent a significant reduction in the "paperwork burden" for both the licensee and the regulatory agency. Also, in Illinois and several other Agreement States, a nominal fee is charged for this particular type of general license.

Cc *,.*

Add I i

1 :*()M/v\\lSSIO

,'. ".'CCTION

~->~i \\ RY I

iZ-

Secretary of the Commission Page 2 March 21, 1988 Second, the Department recommends that the general license for this application be listed separately from the general license granted in 10 CFR 31.ll(a). The current general license for in-vitro use of radioactive material is directed at hospitals and clinical laboratories. Inclusion of phosphorous-32, as petitioned, would permit food laboratories to also possess the other radioactive materials l isted in 10 CFR 31.ll(a). This was not the intent of the original regulation or of the petition.

If you have any questions about these comments, do not hesitate to contact Paul Eastvold, Manager, Office of Radiation Safety, at {217) 785-9918.

~

Lash TRL:gs

RE P L Y T O A TT N OF:

SU B JECT:

TO:

AFOMS/ SGPR DOCKET NUMBER PETITION RULE PRM -3 /-"'f @

DEPARTMENT OF THE AIR FORCE {53,t:,e 2.~°3)

HEADQUARTERS UNITED STATES AIR FORCE

°7

[JQ(, ri,- -,r, BOLLING AFB DC 20332--6188

!J-jNt'c--

1 O MAR 1988 "88 HAR 14 Al 1 :52 BROOKS A FB. TEXAS 78235-5000 Of t*

rlCt.).:r S~ vhr.iA" '(

GENE-TRAK Systems: Filing of Petition for Rulemaking (Docket No. PRM-3 l~'z_~Kf 'J~~NC~ERVlt.f U.S. Nuclear Regulatory Caianission Washington. D.C. 20555 Attn: Docketing and Service Branch

1.

Although the proposed petition addresses use of this process by food manufacturers and food laboratories, we have an interest in any potential regulatory requirements established for users of this procedure since we foresee potential applications within the Air Force by clinical pathology laboratories in identification and control of transmission of the disease organisms which the petitioner claims can be identified by this method.

2.

Unfortunately, the information provided in the subject notice is not sufficiently detailed in the description of the procedure to allow a full assessment of the benefits or risks of the petitioned regulatory position. No mention is made of expected user exposures or the radiation safety procedural instructions/controls to be provided by the manufacturer.

The following is therefore provided based only upon the data given in the petition and some simple assumptions about the use of the procedure.

3.

Use of 0.5 microcuries of phosphorus-32 per test could yield an expected quarterly extremity dose of 13 rems, clearly in excess of the 25% personnel monitoring level (4.687 rems) as specified in 10 CFR 20. 202 and 20.101.

This is calculated assuming: (1) that the 0.5 microcurie per test is handled for one hour per week: (2) hand exposures occur while handling the radioactivity in thin wall glass vessels or equivalent (Direct skin contamination is not included): (3) the dose rate from 1 millicurie of phosphorus-32 over 1 square centimeter of skin. at the surface of the skin, is 2000 rads/per hour (NEN published data).

The dose rate then for 0.5 microcurie would then be 1 rad per hour (Quality Factor of 1).

4. With the levels of activity associated with a single 75 microcurie vial as implied. we would expect users to have to conduct package surveys: have available radiation detection instrumentation for contamination monitoring:

conduct personnel monitoring: and possibly make assessments of internal dose potentials.

These are health physics considerations which are not normally required by users under the general license of 10 CFR 31. 11.

Based upon the above calculations and these anticipated health physics requirements. we are reluctant to recaamend NRC include this procedure with those now generally licensed under 10 CFR 31.11.

5. It is also unclear how the quantity authorization under 10 CFR 31.11 would be managed.

Will the 100 microcurie authorization of Phosphorus-32 under the MAR 2 1 1988

dlit.L,c,80 by C.:Hl1.,,.****,.,, **** "

oc. ( '

It., t J 1---

1-=- ~...,-IV1

,z, I oij£ f

)

, s *n I I!'

general license be included in the total inventory possession limit of 200 microcuries for Iodine-131. Selenium-75. Iodine-125. and/or Iron-59 as stated in 10 CFR 31.11 (c)(l). or will the P-32 authorization be separate as for tritium and carbon-141 LAWRENCE DONOVAN. Maj

USAF* BSC USAF Radioisotope Committee Office of the Surgeon General cc:

HQ USAF/SGPA I

bOCKET NUMBER PtflTION RULE PRM ~/-,4 (!}

( 5 3 F/2. ~ r.:;-v OO(;K[f((,

USNHC Marvin I. Lewis

aa FEB 24 P 3 :25 Secretary of the Commission USNRC Washington, D. C. 29555

Dear Mr. Secretary:

7801 Roosevelt Blvd.#62 Phila., PA 19152 Please accept this letter as my comments on 10 CFR 31,Docket No. 31-4, Gene Trak Systems: filing of a petition for rulemaking.

What GeneTrak is asking for is an exemption to allow unlicensed food laboratories to store up to 100 microcuries of

wsphorus 32. 100 micrc,curies does nc,t sound like a lot, but it very biologically active and will be in use near food products.

The recent problems with polonium 210 getting into food products from static eliminators should raise a warning beacon.

The food industry does not have great expertise handling radioactive sources. Food technologists are not trained health physicists. Handling radioactive sources is a different technology from food processing. Asking food processors to handle radioactive sources proved to be anathematic with 3M static eliminators. The NRC need not repeat this mistake for Phosphorus

32.

I respectfully urge that the NRC show regulatory restraint.

There is just a lack of need and justification to store phosphorus 32 near food. The test referred to in the petition for rulemaking can be done at an approved laboratory remote from the food processing facility. Also the reputation of the NRC would be 9

tter served if this petitic*n was rejected.

Enclosure:

Staff Suspends Distribution of Static Eliminator devices which use rad ic,act i ve polonium. January 25, 1988.

Mc:ffV in I. Lewis 7801 Roosevelt Blvd.#62 Phila., PA 19152 2-20-88 MAR 1 1988 Acknowledged by c-titcf.* -..,..,..;..~ --..,;;;.,.fl6-** a,-

~ -~ - ~ l'~,.{

~fi';l'Jt'A~Jf "~1M-Ml5'CB

~

tr~.~!3~ &' 5teN-.1~ ~ON!

~IC( C,)r TT-If St~!W~

-i.f *1-* _: ~~.-\\Ml~'.l~t z./~-~- -

1.,.= ----

,:.*d 2,..,

~-=---

f,;s, /!~

14 No.

I-88-7

Contact:

Karl Abraham January 25, 1988 NRC STAFF SUSPENDS DISTRIBUTION OF SOME STATIC ELIMINATION DEVICES WHICH USE RADIOACTIVE POLONIUM The U.S. Nuclear Regulatory Commission staff has issued an order to Minnesota Mining and Manufacturing Company (3M) suspending the distribution of some static elimination devices which use radioactive polonium until the cause of leakage has been determined and corrective action has been approved by the NRC.

The order was issued after Ashland Chemical Company reported that radioactive contamination from the devices has been found at its facilities at Easton, PA, and Dallas, TX.

Ashland reported that the leased devices from 3M are used as air cleaning units to remove dust particles from chemical packaging bottles. Ashland has reported that its radiation consultant at the Easton plant surveyed clothing worn by workers offsite and their automobiles and no contamination was found. Ashland facilities at Newark, CA, and Columbus, OH, were checked by the Company and no contamination was reported.

Both Ashland and the 3M Company are cooperating with the NRC in responding to this incident.

Ashland also said that it will test employees at its Dallas and Easton facilities for internal contamination.

According to the 3M Company, the radioactive polonium is bound in highly insoluble non-respirable particles.

Polonium emits alpha radiation which will not penetrate the skin.

The NRC dispatched a five-member augmented inspection team to the Easton, PA, facility on January 22, 1988.

Two NRC inspectors are at the 3M facility in Minnesota.

The contamination at the Dallas facility is being checked by the Texas Bureau of Radiation Control, since Texas has regulatory authority over that facility.

In its order, issued this morning, t he NRC staff directed the Minnesota Mining and Manufacturing Company, St. Paul, Minnesota, to:

1.

Suspend distribution of some models in the series 900 polonium static elimination devices until the cause for leakage has been determined and corrective action is approved by NRC.

This was the type of device used at the Easton and Dallas facilities.

(more)

15

2.

Immediately inform present users of the devices of the incidents along with instructions to report to 3M if there is reason to suspect that similar problems might exist with devices in their possession.

3.

Immediately start testing of other devices in order to develop a comprehensive test plan which will provide a high degree of assurance that any other leaking devices wi l l be identified.

4.

Provide a technical analysis of the causes of the problem and the way to correct it to the NRC's Region III Administrator at Glen Ellyn, IL.

No. I-88-8 February 1, 1988

Contact:

Karl Abraham NOTE TO EDITORS AND STATION ASSIGNMENT EDITORS Curtis J. Cowgill, Chief of the Reactor Projects Section that inspects Three Mile Island, Unit 1, has issued a status report summarizing activities of the inspection staff during the period December 18, 1987 to January 19, 1988.

The report is attached.

Docket No. 50-289 MEMORANDUM FOR:

FROM:

SUBJECT:

January 25, 1987 Lee H. Bettenhausen, Chi ef, Projects Branch No. 1, Division of Reactor Projects Curtis J. Cowgill, Chief, Reactor Projects Section lA TMI-1 STATUS REPORT FOR THE PERIOD DECEMBER 18, 1987 - JANUARY 19, 1988 Enclosed is the status report from the NRC Resident Office at TMI-1 for the subject period.

These reports are an NRC Region I initiative to provide NRC management and the publ ic with highlights of significant events at TMI-1 from an NRC regula-tory perspective.

Your attention is specifically directed to NRC staff reorganization activities as described in the enclosure.

(original signed by)

Curtis J. Cowgill, Chief Reactor Projects Section lA (more)

DOCKET NUMBER.

/

P

. NP P ".3 -4

( S-.J ;=e, Al'~-~)

r759qrdt"ktrrn NUCLEAR REGULATORY COMMISSION USNHC 10 CFR Part 31

~ JAN 28 P3 :1a

[Docket No. PRM-31-4]

OFFJC[ r,;

~ ~ '* t -

DOCK,-- J,

-

1..,,.*

rl, t1. (

LI ING

'Ti' ' **er GENE-TRAK Systems; Filing of Petition for Rulemak ing 8RANC~

v,..

AGENCY:

Nuclear Regulatory Comnission.

ACTION:

Notice of receipt of petition for rulemaking from GENE-TRAK Systems.

SUMMARY

The Conmission is publish ing for public comnent this notice of receipt of a petition for rulemaking dated November 18, 1987, which was filed with the Conmission by GENE-TRAK Systems.

The petition has been assigned Docket No. PRM-31-4.

The petition requests that the Cormiission establish that 100 microcuries of phosphorus-32 used in GENE-TRAK Salmonella and Listeria assays by a food laboratory is an exempt quantity under a general license according to §31. 11.

DATE:

Submit comments by (60 days after publication in the Federal Register).

Connents received after this date will be considered if it is practical to do so, but assurance of consideration cannot be given except as to conments received on or before this date.

ADDRESSES:

All persons who desire to submit written cormnents concerning the petition for rulemaking should send t heir conments to the Secretary of the Commission, U.S. Nuclear Regulatory Connission, Washington, DC 20555, Attention: Docketing and Service Branch.

For a copy of the petition, write the Division of Rules and Records, Office of Adm1n1strat1on and Resources Management, U.S. Nuclear Regulatory Colffll1ssion, Washington, DC 20555.

The petit1on and copies of collll!ents may be inspected and copied for a fee at the NRC Public Document Room, 1717 H Street, NW., Wash1ngton, DC.

FOR FURTHER INFORMATION CONTACT:

David L. Meyer, Chief, Rules and Procedures Branch, D1v1s1on of Rules and Records, Office of Admin1strat1on and Resources Management, U.S. Nuclear Regulatory Collll!iss1on, Washington, DC 20555, Telephone:

301-492-7086 or Toll Free: 800-368-5642.

SUPPLEMENTARY INFORMATION:

Background

The food industry and its regulators are continuously concerned with the transmission of disease producing organisms, or pathogens, through processed food products. Food laborator1es throughout the country routinely test food products for the presence of pathogens such as Salmonella and Listeria.

Salmonella, an 1mportant cause of food transmitted illness, 1s widely distributed in nature. There are approximately 2,000 different Salmonella organisms all potentially harmful to humans. Because of the widespread nature of Salmonella 1n the environment, there 1s the possibility that food products may be contaminated after processing. Therefore, many food manufacturers routi nely test finished products for the presence of Salmonella.

Listeria is an emerg1ng pathogen of spec1al significance.

In contrast to common food poisoning agents which general ly cause gastrointestinal disease, Listeria infection can produce severe disorders such as meningit1s, septicemia, and abort1on.

Lister1a, which 1s ubiquitous in nature and is able to survive and multiply at refrigeration temperature, is a contaminant most likely to be found in foods of animal origin. The presence of species of Lister1a may be s1gn1ficant indicators of food plant sanitation conditions.

The Petitioner GENE-TRAK Systems is a joint venture formed between Integrated Genetics, Inc., Framingham, Massachusetts and AMOCO Corporation of Chicago, Illinois.

The company currently markets and sells in-vitro diagnostic tests for food bacteria such as Salmonella and Listeria in food products.

GENE-TRAK Systems has applied new technology to improve on conventional microbiological methods of identifying these pathogens.

The Process Because of new advances in molecular biology, it is now possible to isolate DNA from the bacteria to be tested for, label it with phosphorus-32, a rad1oactive isotope, and use 1t in a test system to identify the bacteria 1f it is present in food samples.

The petitioner has developed test procedures using DNA probes for both Salmonella and Listeria. The petitioner asserts that these tests are more accurate because the DNA probes are highly specific.

In addition, the tests are speedier, requiring only two days for completion rather than the seven days needed for conventional methods.

The Problem Because of the presence of byproduct material in the form of phosphorus-32 in amounts exceeding currently exempt quantities, those desiring to use the DNA probe assays must apply for and obtain a specific license from the NRC that authori zes the use.

The amount of phosphorus-32 used per test is 0.5 microcurie and the amount shipped in a single vial is 75 m1crocur1es.

The assays are in-vitro diagnostic tests conducted in food laboratories.

The Sol ution The petitioner requests that NRC amend §31. ll(a) to include food laboratories and to include up to 100 micorcuries of phosphorus-32 as an exempt quantity.

The petitioner asserts that authorizing the use of these products under a general license would assist food manufacturers and food laboratories by eliminating the licensing procedure.

In addition, the paperwork burden on both the NRC and the industry would be reduced.

Dated at Washington, DC this ).1 '!,iay of January 1988.

For the Nuclear Regulatory Conmission.

Secretary f the Conm1ssion.

31 New York Avenue

= GENE-TRAK sYSTEMS Framingham, Massachusetts 01701 DOCKETED USN[c_:fgt i 72-3113 R ILE~ _g_R~f~E-1.J --

DOCKETED USNRC DOCKET NUMB PETITION RUL£ PR~ -

(£1~,I/,£~

a? NOV 23 ~iO :59 fl/J. 11-.]/ - 'I W tlJV 25 P3 :09 November 18, 1987 Director, Division of Rules and Records Office of Administration U.S. Nuclear Regulatory Commission Washington, DC 20555 Attn:

Chief, Rules and Procedures Branch RE:

Petition for Rulemaking To Whom It May Concern:

GENE-TRAK Systems, a joint venture formed between Integrated Genetics, Inc., Framingham, MA and AMOCO Corporation of Chicago, IL, is petitioning for a rule change.

The company currently markets and sells in-vitro diagnostic tests to food company laboratories.

The test kits are used to detect the presence of pathogenic bacteria such as Salmonella and Listeria in food products.

Food-borne pathogens have long been a concern within the food industry, by the U.S. Food and Drug Administration, and by the U.S.

Department of Agriculture.

In 1985, an outbreak of salmonellosis caused over 15,000 people to become sick in Illinois. Also in the same year, an outbreak of listeriosis in Los Angeles resulted in the deaths of 85 people.

Recognizing the need to improve upon the conventional microbiological methods to identify these pathogens, GENE-TRAK Systems has applied new technology to improve these tests. Recent advances in molecular biology have made it possible to isolate DNA from the bacteria of choice, label it with 32 phosphorous, and use it in a test system to identify the bacteria should it be present in food samples.

The key advantages of this approach are improved accuracy, since the 11DNA probes 11 are highly specific, and speed.

These new tests can be completed in two days rather than the required 7-14 days for conventional methods.

Director U.S. Nuclear Regulatory Commission November 17, 1987 page 2 Many food laboratories across the country, as well as U.S. FDA and USDA laboratories, are either using these tests, or are in the process of obtaining a specific license to use these tests.

The amount of 32 phosphorus isotope used per detemination is 0.5 microcurie, and the amount shipped in a single vial is 75 microcuries. These in-vitro diagnostic tests are conducted in food laboratories and it is for this reason we are petitioning for the following rule change.

GENE-TRAK Systems is hereby submitting, in accordance with 10 Code Federal Regulations Part 2802, a Petition for Rulemaking.

The Petition for Rulemaking is being submitted to amend 10 CFR

31. 11 (a) and 31. 11 (a) (7), respectively.

Specifically, Section 31.ll(a) is being amended to include food laboratories under this provision and Section 31.ll(a) (7) to include up to 100 microcuries of phosphorous-32 as an exempt quantity.

It is our opinion that the information as submitted supports the action as sought by this petition.

If you have any questions, please contact me at (617) 872-3113.

Sincerely yours,

~~

Karenlee Voltz-Dow Manager, Regulatory Aff s

/1270a

PETITION FOR RULEMAKING GENE-TRAK SYSTEMS

ATTACHMENT A

B C

D E

F PETITION FOR RULEMAKING GENE-TRAK SYSTEMS INDEX OF ATTACHMENTS CONTENTS Introduction Profile of GENE-TRAK Systems Listeria - Background GENE-TRAK - Listeria Assay Salmonella - Background GENE-TRAK Salmonella Assay

PETITION FOR RULEMAKING GENE-TRAK SYSTEMS ATTACHMENT A Introduction

INTRODUCTION PETITION FOR RULEMAKING GENE-TRAK SYSTEMS Salmonella is probably the most important cause of foodborne illness on a worldwide basis.

The organism is widely distributed in nature, the most important reservoirs being wild and domestic animals.

There are approximately 2,000 different Salmonella serotypes; all serotypes are considered potentially pathogenic for man.

Consumer mishandling of food products is a major contributing factor to the salmonellosis problem in the United States. Considering the widespread nature of Salmonella in the environment, there also -

exists the possibility of post-process contamination of processed food products.

For this reason, many food manufacturers routinely test finished products for the presence of Salmonella. Other important contributing factors in human salmonellosis include cross contamination between raw and cooked products and improper cooling and storage of cooked food.

Listeria is an emerging foodborne pathogen of special significance since, in contrast to common food poisoning agents which generally cause gastrointestinal disease, infection with Listeria can produce severe disorders such as meningitis, septicemia and abortion.

At this time, only..!.._,_ monocytogenes is of regulatory significance from the perspective of FDA, although other species of Listeria have possible importance as indicators of food plant sanitation conditions.

The GENE-TRAK Assays have proven to be faster and more accurate in detecting bacterial pathogens in foods, thus reducing the possibility that contaminated food will be released to the market.

Our customers currently must apply for a specific license to use microcurie amounts of phosphorus-32 which is incorporated in the GENE-TRAK Salmonella and Listeria Assays.

The in-vitro diagnostic tests are used to detect the presence of these pathogenic bacteria in food products.

The revision of the regulations to authorize the use of such a product under a general license would assist both food manufacturers and food laboratories by expediting the license procedure and reduce the paperwork burden on both the NRC and industry.

The revision in the regulation is urgently needed as based on the fact that there may be a human health issue involved.

127Oa

PETITION FOR RULEMAKING GENE-TRAK SYSTEMS ATTACHMENT B Profile of GENE-TRAK Systems

SUPPLIER PROFILES GENE-TRAK Systems GENE-TRAK Systems is a joint venture formed between Integrated Genetics, Inc. of Framingham, MA, and Amoco Corporation of Chicago, IL. The mission of GENE-TRAK Systems is to develop and market nucleic acid probe-based diagnostic products for food and clinical laboratories. Probe technology is an advanced diagnostic procedure that al-lows the rapid detection of disease causing microorganisms.

GENE-TRAK Systems' first product, a DNA hybridization assay for rapid detection of Salmonella in food products, was introduced in 1985. The product reduces test-time from 5 days to less than 48 hours5.555556e-4 days 0.0133 hours 7.936508e-5 weeks 1.8264e-5 months , thus lowering expensive storage costs to food companies.

In 1986, following extensive comparative and collaborative studies, the AOAC unani-mously approved Official First Action. Based on data generated during testing, the GENE-TRAK Salmonella Assay proved to be the most accurate method currently avail-able for detection of this pathogen. 1 The technology under development by GENE-TRAK scientists involves nucleic acid probes, fragments of DNA or RNA specific for an organism of interest which, when la-beled with an identifiable "tag," can be used to determine the presence or absence of a particular organism in a test sample. Key patents surrounding this technology are cur-rently held by GENE-TRAK Systems. This probe technology is based on the fact that nu-cleic acids are comprised of a sequence of nucleotides that is unique for each organism.

For a particular target organism, a nucleic acid probe of complementary sequence can be produced and used in a hybridization reaction in which the probe sequence pairs with its complementary target nucleic acid if that organism is present in the test sample. Such a probe has been developed by GENE-TRAK Systems and is already in use for Salmo-nella identification. The probe has been tested against a large panel of Salmonella iso-lates representing all major serotypes and has been proven to be specific without cross-reacting with other biochemically similar enteric bacteria.

In mid-1986, the GENE-TRAK Campylobacter Assay was announced. This new test is currently undergoing field trials for use in both food and clinical samples and is expected to be introduced in early 1987. Several other nucleic acid-based diagnostic tests for food-borne pathogens are in development and planned for future release.

GENE-TRAK Systems has succeeded in developing these new technologies because of its skilled and dedicated staff. The Company has attracted an outstanding research group with significant experience in pure and applied science. The management team has wide experience in both start-up ventures and work with major corporations in the diagnostics market. The present staff of over 50 represents one of the largest groups in the world dedicated to nucleic acid probe diagnostics, and major expansion is underway.

While recruiting and supporting key people is paramount to a successful venture, ade-quate financing is also essential.

GENE-TRAK Systems plans to invest over $20 million during the next two years to de-velop and commercialize additional diagnostic products. This level of funding is expect-ed to greatly facilitate research, development, and marketing of exciting new diagnostic products based on the continued advances being made in nucleic acid probe technol-ogy.

1 Based on comparison of false negative results.

GENE-TRAK SYSTEMS Reprinted From FOOD ENGINEERING, January 1987

l ~

GRATED

"='GENETICS FOR IMMEDIATE RELEASE

Contact:

Patrick J. Connoy Integrated Genetics (617) 875-1336 Meta Wagner Barbara Kalunian Cone & Company (617) 227-2111 INTEGRATED GENETICS AND AMOCO ANNOUNCE JOINT VENTURE TO COMMERCIALIZE PROBE TECHNOLOGY NEWS Carl F. Meyerdirk Amoco Corporation (312) 856-5566 FRAMINGHAM, MA. July 29, 1986 -- Integrated Genetics, Inc., Framingham, Massachusetts and Amoco Corporation of Chicago, Illinois today announced the signing of an agreement establishing a joint venture to develop and market probe-based diagnostic products.

Both companies have been actively involved in the research and development of nucleic acid probes over the past five years and the combination is expected to accelerate and broaden the commercialization of a new generation of diagnostic tests for the food microbiology and clinical diagnostic markets.

The Joint Venture plans to invest in excess of $20 million over the next two years to develop and commercialize its products.

Located adjacent to Integrated Genetics' facilities in Framingham, the Venture will be staffed by personnel from both Integrated and Amoco.

The newly formed Venture represents one of the largest groups in the world dedicated to the commercialization of this exciting new technology.

-more-31 NEW YORK AVENUE, FRAMINGHAM, MASSACHUSETTS 01 701 617-875-1336 TELEX: 948654 INGN

INTEGRATED GENETICS/AMOCO PAGE THO In addition to contributions of staff and technology by both companies, Integrated Genetics will transfer its food diagnostics business into the Joint Venture including its GENE-TRAK8 DNA probe-based test for Salmonella bacteria in food and its recently introduced test for Campylobacter bacteria in clinical specimens.

Amoco will provide funding to the Venture in the form of working capital for operations, including licensing fees and royalties.

Under the terms of the agreement, Integrated Genetics will be the managing partner with its current President and Chairman of the Board, Robert J. Carpenter, acting as Chief Executive Officer of the Joint Venture.

Integrated 1 s Vice President of Sales and Marketing, Patrick J.

Conney, has been appointed Vice President and General Manager.

According to Mr. Carpenter, uThis Joint Venture with Amoco Corporation is the most significant business arrangement in the history of our Company and should maintain our leadership position in t~e development and marketing of probe-based diagnostic products.

Both companies have made major commitments to probe research and development and the Joint Venture is an ideal vehicle to ensure our mutual success.

Our expertise in isolating probes for a variety of orga~isms and designing practical test formats along with our proven ability to successfully market these products fits well with Amoco's technical expertise in sample preparation, detection, and supporting instrumentation development.

11 Dr. Edward A. Mason, Amoco's Vice President of Research and Development,

stated, 11The formation of this Joint Venture is the key to commercializing an important new technology which Amoco has been actively pursuing in our research facilities for many years.

Integrated Genetics*

demonstrated success in bringing practical probe-based tests to market makes them an attractive partner in achieving our mutual goal of building a major diagnostics company.

The combtnation of our R&D groups, patents and proprietary technologies and the contributions of key scientific consultants for both companies should assure the Joint Venture a leadership position in exploiting the potential of this new diagnostic technology.

11

-more-

INTEGRATED GENETICS/AMOCO PAGE THREE Amoco Corporation is one of the largest petroleum and chemical companies in the U.S.

The corporation has major interests in oil, gas, chemical and other activities in more than 40 foreign countries, and in recent years has been exploring a number of applications of biotechnology.

Integrated Genetics is a leading biotechnology company that develops, manufactures and markets diagnostic and therapeutic products utilizing proprietary techniques based on recombinant DNA technology.

The company specializes industries.

INGN.

in the development of products for the health care and food Its stock is traded on the over-the-counter market symbol

PETITION FOR RULEMAKING

\\

GENE-TRAK SYSTEMS ATTACHMENT C Listeria - Background

LISTERIA FACT SHEET oescript1on Listeria is an emerging foodborne pathogen of special significance since, in contrast to common food poisoning agents which generally cause gastrointestinal disease, infection with Listeria can produce severe disorders such as meningitis, septicemia and abortion. Of tha seven recognized species of Listeria, only one*,,L.

monocytogenes, has been implicated as a foodborne pathogen. At this time, only.L. monocytogenes is of regulatory significance from

,e the perspective of the FDA, al~hough the other species of L1ster1a have possible importance as indicators of food plant sanitation conditions.

Where Present Listeria is widely distributed 1n nature. Fecal carriage is common*

among wild and domestic animals, and has been reported in healthy humans as well. The organism has also been isolated from soil, improperly fermented silage, and leafy vegetables. Historically, lister1os1s has been considered primarily a disease of animals, chiefly livestock. Events within the past five years have 1ffi.p11cated Listeria as a significant cause of foodborne disease in man.

Of particular importance from the viewpoint of the food sanitarian is the organism's ubiquitous presence in nature and its ability to survive and multiply at refrigeration temperatures.

These characteristics stress the importance of prevention of post-process contamination of food products.

Foods of animal origin. including meat, poultry. and dairy products. are those most likely to be contaminated with Listeria. Standard pasteurization processes are believed sufficient to kill Listeria. although there are some data indicating that milk contaminated with high numbers of Listeria may yield some viable organisms after minimum high-temperature short-time pasteurization. This remains a controversial topic.

c11n1caJ Symptoms In healthy adults, 11ster1osis is most frequently manifested as a self-limiting "flu-like 11 illness. However, infection of pregnant women has severe consequences for the fetus, including neonatal sepsis or meningitis, often resulting in abortion, stillbirth, or delivery of acutely 111 infants.

Irmiunocompromised adults constitute a second susceptible population. Mortality in diagnosed cases is on the* order of about 3ot, with mortality occuring most frequently among newborns and patients over 70 years of age.

foodborne Outbreaks In recent years, a number of foodborne listeriosis outbreaks, with high mortality rates, have been reported. Incriminated products were coleslaw, pasteurized milk, and soft cheeses.

The most well-publicized outbreak occured in Southern California in 1985, in which soft Mexican-style cheese was identified as the vehicle of infection. Approximately 200 cases of listeriosis were identified, with a mortality rate of about 4-0i.

Conventional Testing Methods Current tesUng*methods for identification of Listeria in foods depend upon basic microbiological procedures including cultural enrichment and biochemical identification. The method in widespread use for the analysis of dairy products, the 11 FDA method". requires a minimum of 9 days of analysis time before a sample can be identified as Listeria - free. Identification of positive samples requires approximately 7-18 days of analysis time. Another culture method has been developed by_the USDA for the analysis of raw and processed meat products.

GENE-TRAK Listeria Assay The GENE-TRAK Listeria Assay utilizes highly s'pecific DNA probes to identify the presence of L1ster1a species in dairy products and environmental samples following an abbreviated cultural enrichment procedure.

Listeria-free samples can be identified within 48 hours5.555556e-4 days 0.0133 hours 7.936508e-5 weeks 1.8264e-5 months . Confirmation of positive samples is accomplished by standard cultural techn~ques.

In addition to the obvious benefit to the food producer of decreased analysis time and therefore earlier product release, preliminary results of field trials have indicated that the GENE-TRAK Assay is more effective than the FDA culture procedure in identifying the presence of Listeria in test samples.

/2020b

PETITION FOR RULEMAKING GENE-TRAK SYSTEMS ATTACHMENT D GENE-TRAK Listeria Assay

I I

I GENE-TRAK SYSTEMS

I I

I,.

I I

GENE-TRAI{li Listeria Assay A DNA probe hybridization assay for the rapid detection of Usterls species in dairy and environmental samples lf//!IJ. GENE-TllAK ii!!!!E-SYSTEMS 31 N*v York AY*nu*

Framingham., MA 01701

I I

I ---

1 I

I I

I GENE-TRAKm Listeria Assay Abstract Recent documented foodbome outbreaks of listeriosis have underscored the need for improved isolation and identification procedures for Listeria. The application of simplified rapid nucleic acid hybridization techniques, in combination with improved cultural methods, is one of the most promising of approaches today.

The GENE-TRAK Listeria Assay is based on a highly specific nucleic acid hybridization technology that delivers improved accuracy in a rapid, 48-hour procedure. The format of the test is similar to the GENE-TRAK Salmonella Assay which has proven to be an easy, reliable and accurate method for the detection of salmonellae in all major food types.

This assay is designed for the screening of Listeria species in dairy and _

environmental samples. It has a demonstrated specificity for all species of Listeria, and has been studied extensively against a broad range of other microorganisms, including closely related gram positive organisms.

The Listeria Assay provides a 48 hour5.555556e-4 days 0.0133 hours 7.936508e-5 weeks 1.8264e-5 months test result compared to the 9-14 day FDA method. Preliminary studies have shown a reduced false negative rate relative to the FDA culture method. These data were obtained from two trials conducted by a large, independent reference testing laboratory. Samples included finished dairy pr_oducts and environmental samples routinely submitted for Listeria analysis.

The GENE-TRAK Listeria Assay represents a sensitive and specific alternative to conventional culture methods in the routine detection of Listeria species, and provides test results one week sooner.

I I

I.,

I,.

I I

I I **

I I

LISTERIA Infections:

-Background & Implic3:tions Two related series of events have contributed to an increased interest in improved assays for the identification of Listeria monocytogenes (and other Listeria species) principally in dairy products in the United States and Europe:

A. Significant recent disease outbreaks (e.g., coleslaw, milk, soft cheese) epidemiologically associated with Usteria monocytogenes; product recalls (e.g., ice cream, soft cheeses);

and, product impounds (e.g., imported European cheeses) associated with Listeria contamination.

8. The FDA Dairy Program Initiatives for increased microbiological surveillance,-in light not only of Usteria contamination, but also increased isolation of Yersinia, Salmonella and enterotoxin-producing staphylococci.

RECENT FOODBORNE LISTERIOSIS OUTBREAKS Location Vehicle/mode Cases/deaths Comments Maritimes, Canada Coleslaw/manure 41/19 contaminated, 1981 refrigerated slaw Massachusetts Milk 49/29 listeriosis 1983 in herd California Soft cheese

~200/~100 contaminated plant, 1985 poor pasteurization In the case of Usteria monocytogenes, some of these events have involved deaths, product recalls, and significant legal actions. Hence, the desire for improved testing methods comes primarily from manufacturers and regulatory agencies charged with surveillance and safety responsibilities.

I I

I

I I **

I I

.I*

I **

I I

I I,

In view of this level of interest, it is increasingly important for food microbiologists to understand this organism and the disease.

Listeriosis does not have a long history as a specific disease entity. As late as 1950 only about 100 cases had been described in the medical literature. Listeriosis was first described in rabbits by Murray et al. in 1926, and in man by Nyfeldt in 1929. In rabbits, the infection was associated with a pronounced mononuclear leukocytosis (hence, "monocytogenes"); a pleomorphic gram positive rod, previously undescribed, was isolated from blood and tissues of many animals dying from this new disease. The fact that the organism was observed microscopically, but not always cultured, gave the first indication that L. monocytogenes might require special enrichment and growth conditions.

Most of the early studies concerned disease in animals, particularly sheep and cattle where economic losses due to listeriosis were greatest. The disease in animals was most often manifested as septicemia, encephalitis or meningitis, and neonatal infection or abortion - i.e., the same clinical picture as found in human disease. A primary goal was to establish the epidemiology and pathophysiology of the disease. Therefore, much effort went into isolating Listeria from environmental sources, soil, silage, feeds, feces, or infected tissues.

All of these samples have high levels of competitor organisms making overgrowth a significant problem. This led to the continuing effort to develop highly selective media and differential culture methods specifically for these specimen types, i.e., not optimized for common food samples.

The major studies of human listeriosis, and relationships between animal and human disease began only in the early 1950's. There was much speculation concerning increased incidence among people associated with animals, but no clear-cut role for a foodborne route was established until a few years ago when outbreaks were related to contaminated coleslaw and milk. Asymptomatic carriage (e.g., fecal or vaginal) is found in about 5% of humans sampled. Clinical or subclinical infection results in production of antibody but this is not helpful diagnostically since:

1) antibody does not appear until late in the course of acute disease;

2) antibodies are frequently found in the sera of healthy individuals;

3) anti-Listeria antibodies may cross-react with other gram positive organisms (especially staphylococci and streptococcij.

I I

I,.

I I

I*

I I

I The most common form of listeriosis in man is either subclinical or a mild flu-like illness that goes unrecognized. Those susceptible to clinical illness include neonates, the unborn, the elderly, and immunocompromised patients. In these groups, septicemia and meningitis contribute to mortality rates of up to 31 %. The CDC estimates that approximately 1 o*oo cases occur yearly in the U.S.

(probably an underestimate).

The extent of the soft cheese outbreak and the conditions present in the manufacturing facility highlighted the potential for other such events.

Hospital-acquired infections (outbreaks?) have also been reported. In 1986 when Listeria was found in several lots of European cheeses (for export to the U.S.), it became clear that the testing effort had to be greatly increased. Very quickly non-specialists became reminded of the many unanswered questions concerning the disease, and the unsatisfactory nature of culturing procedures then available. Increased emphasis was put on developing improved recovery techniques at CDC, FDA, in several academic centers, and in some industrial and reference laboratories. Additionally, trade organizations (e.g., Dairy Research Foundation) began to sponsor symposia on the nature of Listeria, and to support research on better analytical methods, epidemiology of the disease and pathophysiology of the organism. These efforts led to several improved cultural methods.

Seven species are now recognized in the genus Listeria :

L. monocytogenes, L. innocua, L. see/igeri, L. ivanovii, L. welshimeri, L. grayi and L. murrayi. "L. denitrificans " has recently been renamed Jonesia denitrif,cans. All of these organisms are now considered "ubiquitous" in soil and environmental samples. Once jso!ated they all grow very well on ordinary media. Listeria monocytogenes is the only species which is a significant human pathogen (i.e., >99% of cases);

L. ivanovii has rarely been recovered from human infections.

Isolated colonies can be identified on the basis of gram stain results, production of hemolysin, and a variety of biochemical tests, including use of the gram positive identification systems. Finally, isolates can be serotyped and specific serotypes appear to be more commonly associated with disease (e.g., in the U.S. types 1 and 4b are most common). The organism will grow over a wide range of temperatures (including 4°C), does not survive below about pH 5, but can tolerate a variety of toxic chemicals including tellurite, acridine dyes, lithium

I I

I I,,

I 1-. **

I I

chloride, and seleeted antibiotics (e.g., naladixic acid, polymyxin B,

cycloheximide, moxalactam). These materials have been incorporated into dozens of formulations of selective media. Persistence and growth at 4°C is the basis for the very sensitive method of cold enrichment in which a sample is refrigerated and subcultured over a period of weeks to months.

The GENE-TRAK rapid hybridization procedure, with improved culture enrichment, simple format, and objective end-point,. detects all Listeria species. This new approach will provide significantly faster turnaround in Listeria testing with improved accuracy.

I-I I

I I

I I --

1 I

I I

I: **

I I

GENE-TRAK Method Sample Preparation Primary Enrichment Dairy Product samples

1. Add 25 g sample to 225 ml Listeria Enrichment Broth (LES).

Homogenize using blender or stomacher as appropriate for particular sample type. Incubate for 24 +/- 2 hours2.314815e-5 days 5.555556e-4 hours 3.306878e-6 weeks 7.61e-7 months at so0c.

Environmental Samples 1

Place swab, sponge or other environmental sample in flask or bottle containing 25 ml (or larger volume if required)

LES. Incubate for 24 +/- 2 hours2.314815e-5 days 5.555556e-4 hours 3.306878e-6 weeks 7.61e-7 months at so0c.

Secondary Enrichment All Samples

1. Remove the primary enrichment culture from incubation and mix well. Transfer 1 ml of primary enrichment culture to flask or bottle containing 100 ml Modified Listeria Enrichment Broth (MEB-see GENE-TRAK Listeria Assay Instruction Manual for media formulation). Incubate for 24 +/- 2 hours2.314815e-5 days 5.555556e-4 hours 3.306878e-6 weeks 7.61e-7 months at 35°c.

I I

I I **

I I

GENE-TRAK Protocol Overview Part 1. Preparation

1. Enrich food or environmental samples according to the GENE-TRAK protocol.!

2. Set up GENE-TRAK Manifold with sample cups sufficient for the number of tests.

3. Complete the data analysis form.

4. Reconstitute the Enzyme Concentrate with 15 ml of Enzyme Buffer.

Part II. Analysis

1. Add 0.5 ml of Enzyme Solution to a 12x75 mm tube.

2. Add 1 ml of enriched sample to tube. Incubate 15 minutes at room temperature.

3. Add 0.25 ml of Sample Treatment Solution to the combined Sample/Enzyme Solution mixture. Incubate 15 minutes at room temperature.

4. Pour off contents of tube into a filter cup and filter.

5. Add 2 ml of Denaturation Solution. Wait 2 minutes and filter.

6. Add 2 ml of Neutralization Solution./Wait 2 minutes and filter.

7. Add 2 ml of Fixation Solution. Wait 2 minutes and filter.

8. Place filters in conical tube containing 25 ml of Pre-Hybridization Solution. Incubate at 65°c for 30 minutes. Pour off contents,

9. Add 12 ml of Hybridization Solution and GENE-TRAK Listeria Probe. Incubate at 65°C for 120 minutes. Pour off contents.

1 0. Remove unbound Probe with Wash Solution.

11. Remove filters and count.

Part Ill. Data Report

1. Enter counts for positive and negative controls.

2. Enter count for each sample on the data analysis form.

3. Analyze data to determine final results.

Please Note:

If the DNA-hybridization assay is negatjye. no additional testing for the..detection of Listeria Is required.

If the DNA-hybridizati9n assay is positive, culture confirmation is

I.

I I

Ii I **

I I

recommended for Listeria identification. Streak the secondary enrichment culture (MEB culture which has been incubated for 24

+/- 2 hours2.314815e-5 days 5.555556e-4 hours 3.306878e-6 weeks 7.61e-7 months ) to modified McBride Agar (MMA) both undiluted and diluted in 0.5% KOH. Return the MEB culture to incubation at 35°c for an additional 5 days, then strea}< again, if necessary, as described above. For all platings, incubate the MMA plates at 35°C, and observe daily for the presence of typical Listeria colonies. Identify any suspect colonies by standard morpho1ogical and biochemical procedures.

I I

I,.

I I

I **

I I

GENE-TRAKm Listeria Probe INCLUSIVITY DATA Species (number tested)

L. monocytogenes, serotype 1 /2a (17)

L. monocytogenes, serotype 1 /2b (9)

L. monocytogenes, serotype 1/2c (7)

L. monocytogenes, serotype 3a (2)

L. monocytogenes, serotype 3b (10)

L. monocytogenes, serotype 3c (1)

L. monocytogenes, serotype 4a (1)

L monocytogenes, serotype 4b (14)

L. monocytogenes, serotype 4c (2)

L. monocytogenes, serotype 4d (2)

L. monocytogenes, serotype 4e (1)

L monocytogenes, serotype 4ab (2)

L. monocytogenes, serotype 6b (1)

L. monocytogenes, serotype 7

(1)

Dot Blot Test Results L. monocytogenes, serotype unknown (19)

Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive Positive L. lnnocus, serotype 4b (1)

L innocus, serotype-6a (4)

L. innocua, serotype 6b (6)

L. innocus, serotype unknown (2)

L. lvanovli, serotype 5 (5)

L. Ivanov/I, serotype 5 (1)

L. seeligerl, serotype 1 /2a L. seeligeri, serotype 1/2b L seeligeri, serotype 4a L. see/igerl, serotype 4ab L see/igerl, serotype 4c L seeligeri, serotype 6b (1)

(7)

(1)

(2)

Positive Positive Positive Positive Positive Negative*

Positive Positive Positive Positive Positive Positive

I I

I,.

I, I

I I **

I I

Species (number tested)

Dot Blot Test Results L. welshimeri, serotype 6a (4)

Positive L. welshlmerl, serotype 6b (1)

Positive L. welshimeri, serotype unknown (2)

Positive L. gray/ (2)

Positive L. mullayi (3)

Listeria sp., serotype 1 (1)

Listeria sp., serotype 4 (1)

Listeria spp., serotype unknown (4)

TOT AL NUMBER OF LISTERIA ISOLATES TESTED = 139 Positive Positive Positive Positive 138* POSITIVE

L. lvanovii strain 3360 while negative by dot blot hybridization has been found to be positive repeatedly by the GENE-TRAK Listeria Assay

I I

I I

I **

I I

GENE-TRAK11 Listeria Assay EXCLUSIVITY DATA Species Aeromonas sobria Bacillus cereus.

Bacillus subtilis Brochothrlx thermosphacta Cltrobacter diversus Citrobacter freundil Corynebacterium diphtheriae Cotynebacterium equi Corynebacterium xerosls Edwsrdsiella vulneril Enterobacter agglomerans Enterobacter cloacae Jones/a denitriflcans

Klebsiella oxytoca Klebsiella pneumoniae Lactobacillus easel Proteus vulgaris Pseudomonas aeruginosa Salmonella anatum Salmonella arizonlae Salmonella blegdam Salmonella choleraesuis Salmonella dub/In Salmonella lnfantis Salmonella litchfield Salmonella typhl Salmonella typhlmurium Se"atia sp.

Shigella boydii Shigella dysenteriae Shlgella flexnerl Shigella sonnel GENE-TRAK Test Result Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative Negative

I I

I **

I I

Species Staphylococcus homlnls Staphylococcus saprophytlcus Staphylococcus aureus Staphylococcus epldermldis Streptococcus bovis Streptococcus agalactiae Streptococcus lactis Streptococcus faecalls Streptococcus faeclum Streptococcus pnuemoniae Streptococcus mutans Streptomyces globisporus Yerslnia enterocolitica 28 unidentified Gram + bacilli (from food or environmental samples)

Total Number of non-Listeria species tested = 73 GENE-TRAK Test Result Negative Negative Negative Negative Negative NeQatlve Negative Negative Negative Negative Negative Negative Negative Negative 73 NEGATIVE

formerly named L denitrificans,* and now considered as a separate genus (Rocourt et al. 1987. Int. J. Syst. Bact. 37:

266-270).

I I

I f

I I

I **

I I

GENE-TDAK SYSTEMS Key GENE-TRAKm Listeria Assay Features o Accuracy: Greater sensitivity, fewer false riegatives.

o Speed: Test results*in 48 hours5.555556e-4 days 0.0133 hours 7.936508e-5 weeks 1.8264e-5 months rather than 9-14 days.

o Ease-of-use: Proven GENE-TRAK format with numerically coded, ready-to-use reagents.

o Objective test results: Numerical cutoff calculations to discriminate positives and negatives.

o Simple instrumentation o A growing product line of diagnostic products for foodborne pathogens.

I I

I

PETITION FOR RULEMAKING GENE-TRAK SYSTEMS ATTACHMENT E Salmonella Background

SAUIJNELLA FACT SHEET Description Salmonella is probably the most important cause of foodborne illness on a worldwide basis. The organism 1s widely distributed in nature, the most important reservoirs being wild and domestic animals. There are approximately 2,000 different Salmonella serotypes; all serotypes are considered potentially pathogenic for man.

Hhere present Foods of animal origin, including red meats, poultry, eggs, and raw dairy products, are major sources of human salmonellosis.

Salmonellae are very heat sensitive and are readily destroyed by proper cooking of raw foods and pasteurJzat1on of milk.

Consumer mishandling of raw products is a major contributing factor to the salmonellos1s problem in the United States. Considering the ubiquitous nature of Salmonella 1n the environment, there al,so exists the possibility of post-process contamination of processed food products. Thus, a multitude of food products have the potential for Salmonella contamination and ultimately the possibility of being vehicles in foodborne salmonellosis outbreaks. For this reason, many food manufacturers routinely test finished products for the presence of Salmonella. Other important contributing factors in human salmonellosis include cross-contamination between raw and cooked products, and improper cooling and storage of cooked food.

CJ1n1cal Symptoms In foodborne salmonellos1s, the organisms multiply 1n the intestinal tract, producing symptoms including diarrhea, cramps, vomiting, and elevated temperature.

In otherwise healthy individuals, the illness is of relatively short duration and mortality is low.

High risk groups include the very young,

the aged, and individuals with underlying disease and/or a compromised imnune system. In these populations, the disease can be very severe and death may result. Only recently recognized is that salmonellosis may produce serious diseases as sequelae, including rheumatoid disorders, possibly with cardiac involvement.

Foodborne Outbreaks Documented foodborne outbreaks of salmonellosis are not uncommon.

In the past decade, there have been several outbreaks of foodborne salmonellos1s in the U.S. causing hundreds to thousands of cases of illness. Products including cheddar cheese, pasteurized milk and precooked roast beef have been implicated as sources of infection.

One of the more recent and well-publicized outbreaks, involving pasteurized milk in the Chicago area, resulted 1n an estimated 20,000 cases of illness.

Conventional Jesting Methods Conventional methods of analysis of food products for Salmonella contamination involve microbiological culture procedures and biochemical and serological identification. These microbiological procedures generally require four to seven days for complete analysis.

GENE-TRAK Sa]mope]Ja Assay The GENE-TRAK Salmonella Assay utilizes DNA probe technology to accurately and rapidly identify the presence of Salmonella in food products. Total analysis time 1s approximately 48 hours5.555556e-4 days 0.0133 hours 7.936508e-5 weeks 1.8264e-5 months . thus reducing analysis time by two days or more compared to conventional culture procedures. Advantages to the food producer include reduction 1n inventory carrying costs and lessened response time in the event of a contamination problem.

The GENE-TRAK Salmonella Assay also offers increased accuracy.

In independent studies comparing the GENE-TRAK Assay and the most widely used conventional culture procedure, the GENE-TRAK Salmonella Assay has exhibited superiority in detecting Salmonella contamination in food products.

/2019b

\\

I I

I le I

I I

GENE-TRAK I

SYSTEMS I

I I

I.

INSTRUCTION MANUAL GENE-TRAK8 I

SALMONELLA ASSAY I

I I..

I I

.1 042287 I

I

1 I

I I

I INSTRUCTION MANUAL GENE-TRAK8 SALMONELLA ASSAY DNA HYBRIDIZATION TEST FOR DETECTION OF SALMONELLA TABLE OF CONTENTS Page No.

l. Qualifications of User...............................

3

2. Intended Use.........................................

3

3. Biological Principals of the Test....................

3

4. Materials Supplied ***..........*..,..................

4

5. Storage Requirements..........*.............*........

4

6. Precautions..........................................

5

7. General Handling of Test Kit.........................

6

8. Permissible Quantity of 32P for Storage......*.......

6

9. Materials Required but not Supplied..................

6

10. Accessory Equipment Available........................

7

11. Sample Preparation and Enrichment for Raw Meats, Milk.

7

12. Sample Preparation and Enrichment for Other Foods....

7

13. Sample Treatment........... *.........................

8

14. Hybridization Procedure..****.........*.............. 10

15. Data Analysis........................................ 12

16. Negative and Positive Criteria *.....*................ 12

17. Example Calculation..................*..............* 13

18. Limitations of the Test

19. Sensitivity of the Test 14 14

20. Troubleshooting......................................

15*

21. References............................................

16

22. Manufacturer and Distributor.............*..*.....*.. 16

23. Warranty Provisions.**..**.*.........*................ 17 I

i I

I 1*

I I

I 1*

I GENE-TRA~ Salmonella Assay DNA Hybridization Test for Detection of Salmonella Qualifications of User The receipt, acquisition, possession, use and transfer of this test kit containtng radioactive materials is subject to regulation and the user must obtain a specific license of the United States Nuclear Regulatory Commission or of a State with which the Commission has entered into an agreement for the exercise of regulatory authority.

Intended Use The test is a DNA hybridization assay to detect the presence of Salmonella species (spp.) in food products.

Biological Principals of the Test The DNA hybridization test utilizes specific 32-phosphorus ( 32P)-labeled DNA sequences, called probes, which are unique to Salmonella spp.

Membrane filters supported in polypropylene cups are employed which trap bacteria from food products pre-enriched and selectively enriched in accordance with Association of Official Analytical Chemists (AOAC) and GENE-TRAK Systems guidelines. If any Salmonella cells are present in the food sample, they will be bound to the membrane.

In a series of subsequent additions, incubations and filtrations, the bacteria are lysed and the genomic DNA is separated into complementary strands and using a patented procedure, fixed to the filter membrane.

The filters are then removed from the cups and placed in a 50 ml conical tube for the following steps of pre-hybridization and hybridization.

A pre-hybridization solution is added to the filters, blocking any 11 empty 11 sites on the filter membrane.

After incubating and decanting, the 32P labeled probe is added along with hybridization solution to the filter membranes.

If any Salmonella DNA sequences are present on the filter, the probe will selectively bind to those sequences.

Excess probe is removed from the filters by subsequent washings and the filters are removed from the tube and counted in a scintillation or other beta particle counter.

I I

i I

I -

I I

I This assay is a qualitative test to determine the presence of Salmonella spp.

in food samples. A food sample is considered non~reactive for the presence of Salmonella spp. if the counts per minute (cpm) value for its filter does nQ1 exceed the established CUTOFF value for the assay.

A food sample is considered reactive for the presence of Salmonella spp. if

the cpm value for its *filter is greater than the established CUTOFF value for the assay.

As in most diagnostic tests, false reactive results may occur. A reactive food sample may be retested for repeatability. Samples which are reactive should be confirmed by standard plating techniques, followed by biochemical identification.

Materials Supplied GENE-TRAK Solution Set (Cat. No. GT501)

1. 1 240 ml bottle of Solution 1 (Denaturation Solution)

2.

240 ml bottle of Solution 2 *(Neutralization Solution)

3.

240 ml bottle of Solution 3 (Fixation Solution)

4. l 120 ml bottle of Solution 4 (Pre-hybridization Solution)

5. 1 60 ml bottle of Solution 5 (Hybridization Solution)

6. 3 240 ml bottles of Solution 6 (Wash Solution)

NOTE:

Solutions from different lot sets should not be interchanged.

GENE-TRAK Filter Cups (Cat. No. GT601)

1. 112 filter cup assemblies for 96 tests and 16 controls GENE-TRAK Salmonella Probe and Controls Set (Cat. No. GT701)

1. l 1.3 ml vial containing 0.75 ml of Salmonella DNA Probe Solution

2. l 60 ml bottle containing 20 ml of Positive Control Solution

3. l 60 ml bottle of Negative Control Solution GENE-TRAK Manifold Kit (Cat. No. GT801)

1. 1 manifold base with vacuum control valve, 2 tops and 2 trays

2. 12 No. 3 rubber stoppers Storage Requirements

1. Store Solutions 1-6 at room temperature (15°-24°c).

2. Store Positive Control, Negative Control and Salmonella DNA Probe Solution (in Lucite container) between uses at 2°-s0c.

I I

I Precautions

1. Handling of Food Products The food specimens to be tested should be handled and disposed of as if capable of transmitting salmonellosis.

o Do not smoke, eat or store food in the Salmonella testing area.

o Do not pipette by mouth.

o Rubber or disposable plastic gloves sho~ld be worn throughout the testing procedure.

o In case of spills, bench tops and non-autoclavable items should be decontaminated by treatment for several minutes with a 2.5%

solution of sodium hypochlorite or other laboratory disinfectants (e.g. phenolics, iodophors, etc.).

2. Disposal of Infectious Material The preferred method of disposal of contaminated materials, including gloves, pipettes, and filtration cups, is autoclaving for at least twenty minutes c121°C/15 psi wet steam).

3. Handling of Radioactive Material o Do not smoke, eat or store food in areas where radioactive materials are present.

o Avoid direct contact with radioactive materials by wearing disposable plastic or rubber gloves and laboratory coats.

o Do not pipette any radioactive solution by mouth.

o Spills of radioactive solutions should be wiped up immediately and the contaminated waste discarded with solid radioactive waste.

o Store all radioactive materials in properly designated areas.

o Routinely monitor laboratory benches and personnel using survey meters and body dosimeters.

4. Disposal of Radioactive-Material Under current Nuclear Regulatory Co11111ission regulations, low quantities of 32P liquid waste from this assay may be discarded through a selected sink drain.

Some States and municipalities may have more restrictive regulations; consult local authorities.

I I

The spent filters, pipette tips, shipping vial and 50 ml conical tubes contain traces of 32P and must be treated as 32P solid waste.

For disposal, refer to the accompanying Health Physics Material~ Manual.

5. Handling of Filters Filters should be always be handled with clean forceps.

Gloved or ungloved hands may leave deposits on the filters which interfere with the test results.

General Handling of Test Kit These reagents are for in Y.i1rQ diagnostic use.

Reagents should not be used beyond the expiration date. Solutions from different lot sets should not be I

interchanged.

Radioactive material should be stored in a properly designated area. Handle all food samples as if capable of transmitting salmonellosis.

Permissible Quantity of 32P for Storage Laboratories possessing a Byproduct Material License (Specific License) under 10 CFR Section 30.32, NRC Regulations, may not exceed the specified amount of 32P their license allows them to store at any one time.

The receipt, acquisition, possession, use and transfer of GENE-TRAK DNA Hybridizati9n Test Kits for the Detection of Salmonella are subject to a specific license and to other regulations of the U.S. NRC or of a State with which the Commission has entered into an agreement for the exercise of regulatory control.

Materials Required but not Supplied

1. Pre-enrichment medium (see standard methods, ref. l, or equivalent)

2. Tetrathionate (TT), selenite cystine (SC), gram negative (GN) broths (ref. 1)

3. Culture tubes

4. Incubator (35-37°C)

5. Covered water bath (65°C)

6. Disposable pipettes compatible with sample and solution volumes

7. Precision micropipette (100-200 ul range) with disposable tips I

I I

II I

I I

a. Vacuum aspiration flask, tubing, and in-ltne air filter (A "Y 11 connector is optional for operating two manifolds simultaneously)

9. Vacuum source, adjustable from 8 to 20 inches of Hg

10. Forceps

11. Minute timer, preferably 3 channel, for timing intervals of 2 to 120 minutes

12. Absorbent paper

13. Vortex mixer

14. 50 ml polypropylene conical centrifuge tubes

15. Glass scintillation vials and plastic scintillation mini-vial

16. Beta-radiation detection counter

Note: If using a well-type beta scintillation counter Accessory Equipment Available from Integrated Genetics inserts

1. Rack to hold 50 ml tubes and solution bottles in water bath (GT803)

2. GOMCO vacuum pump (Model #402) (GT811)

3. Lucite shield to attenuate beta radiation emissions

4. GENE-TRAK9 Beta Detector (GT881)

5. Survey Meter (Ludlum Model 2) (GT884))

- Note: One-half inch of Lucite effectively shields the 32P.

TEST PROTOCOL Sample Preparation and Enrichent*for Raw Meats (Red Meat, Poultry, Seafood) and Raw Milk.

NOTE:

ALL MEDIA MUST BE PREWARMED TO 35-37°C BEFORE ADDITIONS AND INCUBATIONS.

1. Pre-enrich the food culture in lactose broth.

Incubate for 22 to 24 hours2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months at 35-37°C.

2. Transfer 1 ml of the pre-enrichment broth to 10 ml of TT.

Transfer another 1 ml of the pre-enrichment broth to 10 ml of SC.

Incubate both cultures for 16-18 hours at 35-37°C.

3. Transfer 1 ml of the TT culture to 10 ml of GN broth. Transfer 1 ml of the SC culture to a second 10 ml of GN broth.

Incubate both GN cultures for 6 hours6.944444e-5 days 0.00167 hours 9.920635e-6 weeks 2.283e-6 months at 35-37°c.

Sample Preparation and Enrichment for All Other Food Products NOTE:

ALL MEDIA MUST BE PREHARMED TO 35-37°C BEFORE ADDITIONS AND INCUBATIONS.

I I

I

l. Pre-enrich the food culture in accordance with BAM/AOAC guidelines (1).

Incubate for 22 to 24 hours2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months at 35-37°C.

2. Transfer l ml of the pre-enrichment broth culture to 10 ml of TT.

Transfer another l ml aliquot of the pre-enrichment to 10 ml of SC.

Incubate each culture for 6 hours6.944444e-5 days 0.00167 hours 9.920635e-6 weeks 2.283e-6 months at 35-37°c.

3. Transfer l ml of the TT culture to 10 ml of GN broth. Transfer another l ml of the SC culture to a second 10 ml of GN broth.

Incubate both GN cultures for 12 to 18 hours2.083333e-4 days 0.005 hours 2.97619e-5 weeks 6.849e-6 months at 35-37°C.

Sample Treatment A maximum of 24 food samples (two manifolds connected in parallel to a single vacuum source) may be run with QM Positive and.t.hr..e..e. Negative Control filter cups.

Control samples provided with the kit should be included in each assay under identical testing conditions to those used for test samples.

All pipetting transfers should be made using either a disposable pipette and pipetting bulb or micropipette with disposable tips.

DO NOT MOUTH PIPETTE.

l. Thaw Positive and Negative Control Solutions in a beaker of warm water on initial use.

Store between uses at 2°-0°c.

2. Thaw 32P DNA Probe Solution at room temper~ture on initial use behind a Lucite shield. Store between uses at 2°-0°c.

3. Equilibrate bottles of Solutions 4 (Pre-hybridization Solution), 5 (Hybridization Solution), and 6 (Wash Solution) in the 65°c water bath before use.

4. When Solution 4 has totally d1ssolved, shake and add 25 ml to the 50 ml conical tube.

Place this tube -- the Hybridization Tube -- in the water bath and return the remaining Solution 4 to room temperature storage.

5. Connect the manifold base to a vacuum source (8 to 10 inches of Hg) via a side-arm flask (l-2L). Add a small volume of bactericidal disinfectant to the filtration flask prior to assay.

An in-line air filter should be inserted between the flask and the vacuum source.

6. Place manifold top on base and insert the filter cup assemblies into the openings. A total of 4 cups out of a possible 28 tests (using two manifolds) should be used for controls; one positive and three negative.

(Avoid handling the filters as this may invalidate test results). Insert I

I I

No. 3 rubber stoppers in any unused openings in the manifold top.

7. Invert, shake or gently vortex the Positive Control Solution several times and pipette 2 ml into the Positive Control filter cup assembly.

Record the filter number on the Data Sheet.

8. Invert, shake or vortex the Negative Control Solution several times and pipette 2 ml into each of the.:t.b.r.il Negative Control filter cup assemblies.

Record the filter numbers on the Data Sheet.

9. Vortex or thoroughly shake the two GN broth cultures from each food sample.

Pipette 1 ml from each tube into a single filter cup assembly.

Record the filter number on the Data Sheet.

Repeat for each set of GN cultures to be tested.

10. Apply manifold vacuum.

Using the manifold valve, turn off vacuum after the broth cultures in all the filter cup assemblies have completely filtered. (If the broth cultures do not filter after 2 minutes of vacuum, refer to the Troubleshooting Section in this manual).

11. Add 2 ml -- enough to cover the filter -- of Solution 1 (Denaturation Solution) to each filter cup assembly.

12. Apply manifold vacuum after 2 minutes.

Using the manifold valve, turn off vacuum after Solution 1 has completely filtered through all the filter cup assemblies.

13. Add 2 ml -- enough to cover the filter -- of Solution 2 (Neutralization Solution) to each filter cup assembly.

14. Apply manifold vacuum after 2 minutes.

Using the manifold valve, turn off vacuum after Solution 2 has completely filtered through all the filter cup assemblies.

15. Add 2 ml -- enough to cover the filter -- of Solution 3 (Fixation Solution) to each filter cup assembly.

16. Apply manifold vacuum after 2 minutes.

Using the manifold.valve, turn off vacuum after Solution 3 has completely filtered through all the filter cup assemblies.

17. Snap top and base of each cup apart.

.L!.tl.ng forceps, remove the.:thin membrane filters and place them in the Hybridization Tube prepared in Step

4.

Up to 28 filters -- 4 control and 24 food sample filters -- can be placed in one Hybridization Tube.

Shake the tube gently. All filters should be completely ilTITlersed.

Incubate the filters in the Hybridization Tube for 30 minutes at 6s0c.

I I

I

' I I

I I

18. After the assay has been completed, the filtrate in the vacuum flask should be autoclaved as indicated above and discarded. Manifold bases, tops, and trays should be treated with a disinfectant solution and washed with water.

Do not autoclave these components.

Hybridization Procedure As in working with any radioactive materials, care should be taken to prevent contamination of non-radioactive items with the radioactive 32P labeled DNA probe.

Personnel performing this test should use disposable plastic or rubber gloves, and wear a lab coat or other protective covering.

In addition, absorbent laboratory bench paper should be used in the test area.

For information on radiation safety and disposal requirements, contact:

GENE-TRAK Systems Customer Service Department 31 New York Avenue Framingham, MA 01701 617/872-3113 or 800-338-TRAK The 32P-labeled DNA Probe Solution is shipped in a lucite container.

This container has been designed to be used as a permanent holder for the DNA Probe Solution vial. Also, replacing the luc1te cover on the DNA Probe Solution vial between uses effectively shields 32P emissions.

NOTE:

MAINTAINING THE DNA PROBE SOLUTION VIAL IN THE LUCITE CONTAINER AT ALL TIMES WILL MINIMIZE THE RISK OF A SPILL AND WILL EFFECTIVELY SHIELD 32P EMISSION.

1. Carefully drain the Pre-hybridization Solution (Solution 4) from the Hybridization Tube and discard.

Mix the bottle of Solution 5 (Hybridization Solution) that has been pre-warmed in the 6s0c water bath.

Immediately add 12 ml of Solution 5 to the Hybridization Tube.

Return the remaining Solution 5 to room temperature storage.

2. Vortex or thoroughly shake the DNA Probe Solution. Using a precision micropipette, add the probe to the Hybridization Tube according to the following schedule:

I I

I

NOTE:

DAY*

1 Tue.

2 Wed.

3 Thu.

4 Fri.

5 Sat.

6 Sun.

7 Mon.

8 Tue.

ul of DNA Probe 127 133 140 147 154 162 170 179 Probe is normally shipped on Monday of each week for Tuesday delivery.

Regardless of delivery date, the amount of probe added should be according to the day of the week as indicated in the schedule (e.g., if probe is received on Thursday, 140ul should be used on that day).

3. Dispose of the pipette tip in a container reserved for radioactive waste.

Return the remaining DNA Probe Solution to the 4°c refrigerator.

4. Carefully tighten the cap on the Hybridization Tube.

Gently invert the tube to completely mix the solutions.

Make certain all filters are completely submerged in the solution.

Incubate for 2 hours2.314815e-5 days 5.555556e-4 hours 3.306878e-6 weeks 7.61e-7 months (120 minutes) at 65°c.

5. Carefully drain the solution in the Hybridization Tube into a wa~te container for liquid radioactive waste.

(The radiochemical concentration of the waste is approximately 1.0 uCi/ml of 32P in the molecular form of DNA).

For disposal, refer to the accompanying Health Physics Materials Manual.

6. Immediately add 25 ml of Solution 6 (Wash Solution) that has been pre-warmed in the 65°c water bath. Agitate the filters in the tube by gentle shaking and inversion for at least 10 seconds.

Be sure that all filters are free from the sides and cap of the tube and that all filters are completely submerged in the solution. Incubate for 5 minutes in the 65°c water bath.

7. Gently mix the tube and carefully drain the solution into the radioactive waste container.

8. Repeat Steps 6 and 7 five additional times for a total of six times.

9. Using a forceps, carefully remove the filters from the Hybridization Tube and place them on a clean piece of absorbent paper.

Discard the Hybridization Tube with the solid radioactive waste.

I I

I.

I I

I 1*

I

.1 I

I I

I

10. Using forceps, sort the filters in the following order: (1) Positive Control Filter, (3) Negat1ve Control Filters, remaining food sample filters. Place each f1lter 1n a plastic m1ni-v1al and clean scint1llation v1al (if required for particular beta sc1ntillation counter).

11. Count each f1lter for 30 seconds in a well-type scintillation counter (sc1ntillation cocktail is.Il.Q1 used), and record the results as counts MI mjnute (cpm) on the Data Sheet. Alternatively, count each filter for 30 seconds using the GENE-TRAK Beta Detector (refer to Instruction Manual:

GENE-TRAK Beta Detector).

Data Analysis

1. Determine the average of the 3 Negative Control f1lters. If th1s average value is greater than 500 cpm, Steps 6 and 7 above under 11Hybr1 d1 za t1 on Procedure 11 must be repeated for ill filters. Accept the data after one additional wash even if the Negative Control average is still greater than 500 cpm.

2. The cpm of the Positive Control filter should be at least 5 times the Negative Control average; otherwise technique may be suspect and the assay should be repeated.

3. Add 500 cpm to the Negative Control average.

This sum is the CUTOFF value.

Any filter with a cpm greater than the CUTOFF value is considered reactive for the presence of Salmonella spp.

Negative Criterion A test sample is considered to be non-reactive for the presence of Salmonella spp. if its filter cpm is less than or _e_g_u_a_1 to the CUTOFF value.

Positive Criterion A test sample 1s considered to be reactive for the presence of Salmonella spp. if its cpm is greater than the CUTOFF value.

As in most diagnostic tests, false reactive results may occur. A reactive food sample may be retested for repeatability. Samples which are reactive should be confirmed by standard plating techniques, followed by biochemical identification.

I 1*

I I

I I..

I I

I eGRATED GENETICS Probe lot no.

Probe amount Positive control lot no.

Positive control Positive control Negative control average

FP6253-l

=

170 FP6241-1

=

12141

=

58.6 IF P/N IS LESS THAN 5, REPEAT TEST Sample#

1 CltlOl 2

gt102 3

gtl03 4

gtl04 5

gt105 6

qtl06 7

gt107 8

qt108 g

at109 10 otllO 11 atlll 12 ot112 13 gtl13 14 qtl14 15 qtl15 16 otl16 17 at117 18 ot118 19 at119 20 at120 21 atl?l 22 qt122 23 ot123 24 at124 GENE-TRAK' Date 9/22/86 Negative control lot no.

FP6226-1 ul Negative control

1=

180 cem

2=

227 cem cpm

3=

207 cem P/N Negative control average

=

2Q5 cem Plus constant

+

500 cem Equals cutoff

=

705 cem Filter#

cpm COl 107 CO2 192 C03 368 C04 276 cos 195 C06 311 C07 157 COB 255 cog l5A ClO fi3n Cll 4R4 C1?

210 001 284 D02 345 D03 288 D04 331 D05 251 D06 273 007 309 DOB 15478 nnQ 4B D10 277 D11 108 D12 202

I I

~

I I

I Limitations of the Test As in other sensitive diagnostic assays, there is the possibility that false positives (non-repeatable reactives) may occur for the following reasons:

1. Inadaquate washing of the filters. It is important to follow the wash procedure closely, as this insures greatest sensitivity and reduces the possibility of a false positive result.

2. Addition of too much Probe Solution to the Hybridization tube.

Check for proper use and calibration of the pipetting device used for delivery of the Probe Solution.

Sensitivity of the Test The sensitivity of the test has been repeatedly measured against standard plating techniques.

The sensitivity has been as low as 2 to 3 x 106 cells/ml when 2 ml of the culture has been assayed.

I 1*

I I

I I..

I I

I.

I I

Troubleshooting Problem Nothing filtered:

Some samples filtered, others difficult or didn 1 t filter.

High background cpm (negative control average greater than 500 cpm)

Low signal on positive control (cpm less than five times the negative control average)

Solution Check vacuum source.

Check for a proper vacuum seal between manifold top and ba$e by lifting the corner of top with vacuum on.

Check for unused filter assemblies and replace with No. 3 rubber stoppers.

Check to see that all filter cup assemblies are properly seated on manifold top.

Temporarily increase vacuum to 15-20 inches of Hg.

Re-wash filters.

Increase agitation during washing procedure.

Check for proper temperature of water bath.

Check calibration of micropipet to insure that proper amount of Probe Solution is added.

C~eck for proper temperature of water bath.

Check calibration of micropipet to insure that proper amount of Probe Solution is added.

I I

~

I I

I REFERENCES

1. U.S. Food & Drug Administration.

BacterjoJog1cal Analytical Manual, 6th ed., Assoc. Official Analyt. Chemists, Arlington VA, 1984.

2. Fitts, R. Food Technology 39:

95-102, 1985.

3. Fitts, R. et al. Applied & Environ Microbiol 46:

1146-1151, 1983.

4. Flowers, R.S. et al. J. Food Sci.52: 781-785, 1987.

Manufactured and Distributed by:

GENE-TRAK Systems 31 New York Ave. Framingham, MA 01701 617/872-3113 or 800-338-TRAK I

I I

I,,

I I

I HARRANTY PROVISIONS GENE-TRAK Systems Limited Product Warranty GENE-TRAK Systems warrants that the GENE-TRA~ Salmonella test kit product (exclusive of equipment carrying specific warranties) will conform to GENE-TRAK Systems* specification for a period of one (1) year from the date of purchase from GENE-TRAK Systems.

Should this product fail to conform to GENE-TRAK Systems*

sp.ecifications at any time during this one-year warranty period, GENE-TRAK Systems will replace this product at no additional charge, except shipping charges which shall be the sole and exclusive responsibility of Purchaser. Replacement products will be furnished on an exchange basis and will be new.

All replaced products become the property of GENE-TRAK Systems.

This limited warranty does not include replacement of damaged product resulting from accident, disaster, misuse, abuse, modification and/or alteration of the product.

THE FOREGOING EXPRESS WARRANTY FOR THE PRODUCT SHALL NOT EXCEED THE PURCHASE PRICE OF THE PRODUCT AND IS IN LIEU OF ALL OTHER EXPRESS AND IMPLIED WARRANTIES INCLUDING THE WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICUL:AR PURPOSE.

SUCH WARRANTY IS LIMITED IN DURATION TO A PERIOD OF ONE (1) YEAR FROM THE DATE OF PURCHASE.

'IF THIS PRODUCT OOES NOT CONFORM TO GENE-TRAK SYSTEMS 1 SPECIFICATIONS AS WARRANTED ABOVE, YOUR SOLE REMEDY SHALL BE REPLACEMENT AS PROVIDED ABOVE.

IN NO EVENT WILL GENE-TRAK SYSTEMS BE LIABLE TO YOU FOR ANY DAMAGES, INCLUDING ANY LOST PROFITS, LOST SAVINGS OR OTHER INCIDENTAL OR CONSEQUENTIAL DAMAGES ARISING OUT OF THE USE OF OR INABILITY TO USE SUCH PRODUCT, EVEN IF GENE-TRAK SYSTEMS HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES, OR FOR ANY CLAIM BY ANY OTHER PARTY.

~GENE-TRAK gSYSTEMS 31 New York Avenue, Framingham, Massachusetts 01701 617-872-3113 I

I I

I

- I I

-I

.I I

I

-1

I I

I I

ML23153A044

Contents

Text

REFERENCE:

SUMMARY

Dear Sir:

Dear Mr. Nussbaumer:

Dear Mr. Nussbaumer:

Dear Sir:

Dear Mr. Secretary:

Enclosure:

Contact:

Contact:

SUBJECT:

SUMMARY

Background

Contact:

Navigation menu

ML23153A044

Text

REFERENCE:

SUMMARY

Dear Sir:

Dear Mr. Nussbaumer:

Dear Mr. Nussbaumer:

Dear Sir:

Dear Mr. Secretary:

Enclosure:

Contact:

Contact:

SUBJECT:

SUMMARY

Background

Contact:

Navigation menu

Search