**~** VI 

# .-
.

# r©~:1 7Noveinbt?r, 

### 1997 . 

### NOV 13 1997 

# 'E o 

# z o ::;> 

# o

.Chief,

# New Chemicals Branch Chemical Control Division . 

## .'. Z**~** ....

# }~ Office ofPollution Preventlon and.Toxics U.S. Environmental Protection Agency' 

### 401MS(SW . Washingto[1, DC 

### 20460 .

.. Re:' . letter ofApplication .... .' . .'. . ..... '.. '.

. .with information on B.alJ/yloliquefaciens,requestirtgthis

orgarJism 

# be included.in§.725.420 as a candidate recipient microorganism fortiered¢xemplion' . .

.On Deoember 30, 1994,.Novo Nordisk BibChemNorth America,lno. (Novo Nordisk) submittedto EPA a fetter requesting oonfirmation .that the designation ofd:3aoillussubtilis, as afeoipientorganism**eligibl~** for

. tiered eXemption. included·Bacillus amyloliquefaciens.

Irithe EPA's Final regulation Under.theToxic substances Control Act: .

. ' ASumma,yofthe PublioComments'anq tfle Agenoy's Response, the ,Agency stated that it did 

## hot oons"ider B. amyloliquefaciens to be synonymoiJswithBacillussubti/is**buHttat'~if** the c<Jmmentorbelieves that·B.amyloiiquefacienswarrants an exemption·basedqn the .crlleria EPA used to evaluate 

## 13. subti/is, EPA'invites the commentor, or anyone else, to examine the riskass(;l$smentprepared for exemption orR subtilis, and utilizing the assessment.asa model for the issues important for eligibility. provideif)formation to EPA supporting a separate listing forB:amylo/iquefaCiens aSB recipient mioroorganism under§ 

### 725.420". . I Novo Nordisk is supplying this Letter 

### ofApplication with information on B. al1?yloliqiJefaciens according to § 

### 725.67 and is requesting this organism be included in . § 

### 725.420 as a candidate recipient microorganism for tiered exemption. As stated in EPA's Integrated Risk Assessment for B. .subtilis, B. amylo/iquefaciens has'· been considered a variant of B. subtilis for a long time arid there have been recent reversals in the taxonomic designationof B. amyloliquefaciens B. amyloliquefaciens exemption Nov.O NordiskBioChem , North'**Amerlca~ I~c.**  state Road 1003 Box 576**·fra~klinton.** NC 27525-0576 Tel. 919-4943000. FAX·919-4943450



### . ....**~** 

## .!a- . 

# 'E o Z inthEfliterature and reference

### manual~. Most ofthe commercially-. 0 

## . . ... > · available intermediatetemperature stabilityalpha amylases today are 0

derived from B. amyloliquefaciens l)trains that were once classified as. . Z Rsubtili{and· fJa.ve been used in enzyme .production

### f~rover 50

### ~~"I!'

# .. years. ........ .. . ... .. .. .. . . / 

# 1

. . . EPA

## sugg~sted that; in gemeral, the Agency "expeCtsthatpeUtions to --- · add specific recipient microorganisms· to the list at. 

### § 725.420 will

ideally be**pr~ceded** by seVeral MCANs .before the neCeSS1:jry ·experience with and information on the microorganism have been'

..**~..** accumulated to provide .EPA with a starting point for determining whether the recipient should be listed as a candidate for the tiered. exemption". Although, to date, .Novo Nordisk is aware of no MCANs ..... or PMNsthat have been submiUed:-for B, amyloliquefaciens, we

. submit that because B.. 'amy/o/iquefaciens hashistoticall>! been-··

considered a.**m~mber** of the**speciesB~subti1is;** most all of the information accumulated in the EPA's Final Deci.sion Document: TSCASection 5(H)(4) Exemption .for Bacilfus subtilis(herein after

. ,- . ,..,.' ' . r referred to as "Final· Decision Document") applies to B. amyloliquefacieris, thus, inessence, 

### EPA has already-performed a .. rist<**asse~sment on~.**  amjrloliqiJefaciensand has the required experience with and necessarytnformation on B..amyI6Iiquefaciens to. list it as a recipient fqrthetiered,exemption. . . . .. . .' , . The requirementsJara tetter ofApplication itemized in§ 725.67 list . several 'general provisions thatshould be provided to· Show thatthe requested exemption will not present an unreasonable risk of injuryto health or the environment. Again, because .. B.. subtilisand B. amyloliquefaciens are so·closely· related, .and, .in fact, were technicallyco!1siderecrthesame species untrl**1987~and** because·, like . B. subtilis, B.amy/oliquefacienshasa lorig history of safe use in the

.enzyme. manufactUring andspecialtychemicafs· industries,· the·

information· compiled by EPA on B. subtilis in·. the Final Risk Assessment of! Bacillus subti/is portion of the Final. Decision Document will apply to B. amyloliquefaciens. The specific requirements outlined in §. 725.67 are provided as Attachment I.

. B. amyloliquefaciens exemption 2I**~** III**'~'** 

# o Z 

# o
. . > We look forward to the EPA's granting of the exemption of . 0 B. afnyloliquefaciens from MeAN and TERA reporting. . .. • Z

... 

## .**...~..** Plea$e cont!:lctme by direct telephoneat 

### 919

# 494~3152 or direct fax

# 1~j~. · at 

### 919 494-3420 if you have any. questions or require·· further __ • · information.

Sincerely,.· 

# vllL

· Scott H.Shore, Ph.D.

Regulatory Specialist Enylosures: (2)

•, l**-~** 7 November 1997 \ .**~** 1/1 

# .- 'E o 

# z. 

# . g ATTACHMENT 1- SPECIFIC REQUIREMENTS . 0 

# '.' . '" . ,..., .....**.~** 

# . • z

. . '.**~11** A) Identification and classification.of the microorganism using

# /~)~ available genotYRi.c and RhenotYRicinformati(m .' .

. - I - . . .. • Bacillus subfilis has long been observed to bea heterogeneous taxonomic group (Priest, ;Goodfellow, and Todd, 1981). For many' years,' researchers rec;ognized a distinctive group of 'bacteria within the Bacillus /3ubtilis species which possessed high alpha amylase . activity (8aptist et aI., 1978; Fukumoto,1943aandb; Gordon et aI., .

. 1973; Sneath, 1'986; Welker' and Campbell, 1967b). . In 

### 1943,

Fukl!moto proposed the nar:neBacilfus amyloliquefaciehsfor this. group (Fukumoto 1943 a and b); however, itwasnot until later that B. .subtilis and B. amyloliquefaciens.couldbe differentiateq by.. .classical biochemicaUests. In 198t,lhe qame B. iamy/bliquefaciens was revived and aforrrial publication issued 'which**established~.** 

### 8: amyloliquefaGiens as a distinct species (Priest el aI., 1987): In that '.publi<::ation, Priest et al. state that 

### "B. amyloliquefaciens strai,ris Can be.·.·

diStinguished from B. subtilis strains by

### the inability of most**st~Clins** to hydrolyze DNA'andp,ectin, the failure of the orgariisms to produce aGidfror:n. inuliri, and the, formation .in most '8...amyloliquefaciens' strains of'/ong chains 

### of cells". The authors suggest that· when biochemic,al testsfail 

# to differentiate**species~** DNA,homoiogy should .' produce'a definite identifi<::ation. . DNAs from' strains', 

### of

8: ainyloliquefaciens share less than 25%. homology with DNAs from .

. strains of B. subtilis.·· .

. .,' " "..',', 

### ".) .' . , 

## By Information to. evaluate the relationshiRofthemicroorganism to other closely related microorganisms which have 

# a ,Rotential for adverse effects on health orthe environment . There are se,veraI species of 'the genus Baciilus that are

### k~6wn pathogens. These, include B. 'anthracis'which 'is pathogenic to

. humans and other animais, and B.cereus which is a common Cause

of food pOisoning (Claus and Berkeley, 1986; Norris et aI., 1981): B. thuringiensis, B. larvae, 

### B. lentimorbus,' B. popilliae, and some strains of B. sphaericus are pathogenic to certain insects. Other species in the genus are considered "opportunistic pathogens".

B. amyloliquefaciens exemption 1 J

## .. . ..lIr! III , 

## .-

'0' iii. In a nLimerical classification study using 118 characteristics of 3680 species of·Bacillus, the· species B. thuringiensis and B. .cereus 

## 2 clusteredtogether at 89-92% similarity (Priest et aI., 1988). The 0 

### .. :>

. B.subtifis group, including B.amyloliquefaciens,joinedthe B.cereus .0.

group

### at72~o relatedness: The authorsstated that, although the. Z .. B:

.. amYI?liqU.,e.fac.iens. clu.ster. was d.iS.tinct .. fro.m 

### B..

## ~ubtifis, ?o..

## nsi.ste~t**~~.'** •.

## H.~.·.. '**d~ff~re~~lal.** features.were n?t.**eVlde~t.·** TheTe IS no**d~cUfty** .In / 

# 'Z • distingUishing between. toxln"produclng stmms of· Bacillus and '.. . , B. amyloliqUefacien& . •



### ... ' C) A history ofsafe ,commercial use for the microorganism

.. Baci[fus amyloliquefaciensisone ofthe most widely used bacteria for

thepmductJon of enzymes and specialty chemicals (Harwood', 1992;

. Ingle and Boyer, 1976).'Aunstrup{1979J reports fhafstrains of

B.amyloliquefaciens have been'industltalsources of carbohydrase, · since at least 1913.. In this article; the uncertainty ofthenomenCiafure .

of, the production organism is noted - initially 

### if**wa~caUedBacilliJs**

. mesentericus;then B:subtilis" and now 

### 8. amyJoliquefaciens: Indeed,

· most of the cOrT]meteial Intermediate temperature alpha amylases

..available..today. areqerived from B, 'amyloliquefaciens· (Teagueand

" -,' • • ..... - - - - '- < -' Brumm, 1992). Manyo{**t~ese** strains are derivatives of strains ,originally named B. sUbtilis that have peen used in enzyme production for over 50 years. . ' . . . 8. amyloliquefaciens arso has a long history 

### of safe use in the prOduCtion of pmtease. Aunstrup(H}79) states that "[p]ractlcally all protElase preparations of bacterial origin produced .before 1960 were madefrom this organism": Agaln,these strains Were initially identified as B. subtilis, butate presently reclassified as**B~** amyloliquefaclens.. Dj Commercial uses indicating that the microorganism products· might be subjectto TSCA , . ·TSCA uses 

# of amylases include desizing of,textiles and starch ·modification for sizing ofpaper and industrial ethanol. TSCA uses of proteases include' cleaning aids in**det~rgents** anddehairing and bating inthe leather industry (Erikspn, 1976). J '

, 'E) Studies which indicate potential .for the microorganism to cause adverse**effe~tsto** health orthe environment 

### J:t. \/I 

# .- 'E o 

# z· o , The safety ofB. amyloliquefaciens is addressed ina re'centreview by

## ~' 'de Boer and DideriGhsen, (1991) 'These, researchers report ,that .. "Z

"B.

## ~mY!"**fO/~que"facl,'7,n,s,** ha,s not

## ~ppe,**~red,'.** in,any" qf th,e, , c,it,ed pap,e, f"S 

## lfeJl1,''" " "dealIng with,Bacillus sp. as ,infectiOUS**~rganls~s:'" ~**  se.arch qf 

## /l/J'

databases for references on- B. amylollquefaclensmfectlons' or '" , ,intoxications revealed no such cases". The t'luthors note that this may , largely be due to the fact, that formanyyears.B.subtilisand 'R**amyloliiJllef~c;ens** were ,considered synonymous, provi(jing further evidence' of the nonpathogeniCity ,and, nontoxigenicity of , B. amyloliquefaciens. " , , '

NoVo Nordisk (Anne Sietske de Boer), has recently completed an ,updated literature search on B. amyloliquefaCiens covering alsQ the, • years' after the 1991, puqrtcationc Again, nolfterptufEl citing B: amyloljquefaciens as an: infectiousorgpnism was found. 'A summary 

### aNne results onhis receritliteraturesearch is, ihcluded as Appendi{t.,' " ", " The Final, DecIsion, Do(:Urhent. on R subtifis discusses the environmental, hazards eif B. subtilis to animals, pla,nfs, pnd other j rriicroorgpnisms. , ,',Again; because. of the , close', taxonomical relationship of

### thes~. two species, 'many 'of the Cited references ,on , Rsubtilisappl"}!to B.amylo/iquefaciens als6'(7 CFR 330, et.**se~f.;,** , Alexander, .1977; Claus and Berkeley;**~986;** ',Katz and Demain" '1977; Korzybskiet at, 1.978;L9gan, 1988; Sneath,1986}. ,',

F} Studies which indicate the survival characteristics ofthe microorganism in the environment , , .' .' like B. siJbtilis, 

### 8. amylo/iquetf)ciens isa common saprophyte that , inhabits soils and is thought focontribute tei nutrient cycling ,due to the

variety of enzymes mernbersof thespecies are capable of producing. B. amyloffquefaciens' can produce endospores that make the ,'organism resistant to' heat and desiccation (Claus and Berkeley; 1986). While specific data comparing the survivability cifindustrial and wild-type strains of 'B.amyloliquefaciensare not currently available in the literature, most' all industrial strains' of B. amylo/iquefaciensused for enzyme production are mutants Which are asporogenic. It is highly likely that any released industrial strains would not survive outside of containment as well as their**wild~type** strain counterparts.
R nwndnlinllt?tnrhn'l<;; pyprnntinnREFERENCES, 7 CFR 330, efseg., as amended.**~** III 

### '- 

# '2 o 

# z o ,> "0 'Alexander, M. ,,1977. Introduction to Soil Microbiology: John Wiley 

# • z 

# j'm and Sons, Inc. NewYark. '

,Aunstrup; Knud.1979., Production, isolation, and economics of

extracellular enzymes: AppL Biochent Bioeng. '2:27-69. Baptist, 

### J. N., 

### M. Mandel, and R. J.Gherna. 1978.,,' Comparative zone electrophqresis of enzymes inthe genus Bacillus. Ipt. J. Syst. " ' " Bacteriol. 28:229c244, '

Claus, D,and R C. 

### W. Berkeley: 1986. Genus Bacillus Cohn1872, ", pp.**1105~1139,** 

### lnP. H. A Sneath et al. (eds.), Bergey's Mariuatof Systematic Bacteriology, Vol. 2. Williams andWilkins Co.,'Baltimore;**M~** 

## , ' ' De Boer, 

## A. S. and B. Diderichsen. ,1991. ,Onthe safety ofBacillUs SUJjfi/iS8r)d Bacillus amyloliquefadens: A review. App1. Microbial: Biotech. 36:1-4. 'Erikson, RJ:1976, "Industrii::d applications ofthe bacilH:,A review

and prospect!Js,pp. 406-419. 

## in p. Schlesinger (ed), Microbiology. ,'AmericanSocielyfor Microbiology,**Washington~** DC.

Fukutomo, 

### J. 1943.' Studies on the production ofbaC\erial amylase. '. I. ISolation ofbacteria'secreting pbtentamylase and their distribution; . J. jl.gric. Chem,Soc. Jpn.**,19:487~503.** (InJapanese.) " Fukutomo, J. 1943. Studi,es on the production of bacterial amylase

II. Bacterial and physiological natwe. 

### J: Agric. Chem. Soc. Jpn. 19-

. 634-640. (In Japanese.) , ' ' ,

Gordon, R. E., 

### W. C. Haynes, and C.**Hor~Nay** Pang. '1973. The genus Bacillus. U. S. Department ofAgriculture, Washington, DC, Harwood, Colin R.19,92. Bacillus subtilis and itsrelatives:, molecular biological and industrial

### workhors~s. TIBTECH1 0:247-256. . Ingle, M.B. and E. 

### W. Boyer. 1976. Production of industrial enzymes by Bacillus species, pp.420-426. In D. Schlessinger (ed.) , 4- Microbiology**~** 197.6. American Society for Microbiology, Washington,'

. DC.

Katz, E.and A C. Demain.. 1977. Thepeptide antibiotics of Bacillus:

.Chemistry, biogenesis, andpossible functions. Bacteriol. Rev. 41:449-47.4: Korzybski.

### T.; Z. Kowszyk-Gindifer; and W. Kurylowicz: 197.8: .' Antibioticsisblated from thegenus Bac/flus (Bacillaceae). pp.1529" 1661. In: Antibiotics" Origin, Nature arid Properties, Veil. III..

.American Society of Microbiology, Washington, DC.

Logi:lO,N. A 1988. Bacillus llpecies of medical and veterinary '. importance. 

### J. Me(t Microbicl. 25:157.-165. . . .

Norris,'J. 

## R, R. 

## C. W. Berkeley"N. ALogan, and AG. O'Donnell: ·.1981. The genera Bacillus and$porolactobaciJlus, pp. 1711-1742..

InM.P. Starr etaL {eds:),Th'eProkaryotes: A Hcmdbook on Habitats; · 'Isolation; aridIdentiticationof Bacteria,Vol. 2.**Springer~Verrag,** Berlinc···

.. \ . . Prie.st, 

### F. 

### G., M. Goodfellow,

### L 

### A. Shute, and

## R.C. W. Berkeley.**~987.** Bacillus amylCi/iquefaciens sp. nOvo,. nom, Rev. Int J. . · Bacteriol. 37:69-71. '.

# P~iest; F. G., MGoodfell0w,and C. Todd. 1981, The-genus Bacillus: .

AmimericaJ'analysis. 

### InR. C. W.Berkeleyand M.Goodfellbw (eds.) TheAerobic Endospore-forming Bacteria., AC?demicPress, London.. Priest, F'-G., M. Goodfellow,.andC. Todd. 1988. Anumerical · classification ofthegenus Bacillus,J. Gen.MJcrobiol. 134:1847- .

1882.. ' . . Seki,T.,T.Oshima, and Y.Oshima. 1975, Tax:onomic study of' · Bacillus by deoxyriboflucleic acid-deoxyribonuCleic acid hybridization

. andinterspecitic transformati0ri. Int..J:Syst. Bacterial.· 25:258-270_ Sneath, P_ H. A 1986. Endospore-forming gram-positive rods and cocci, pp. 1104-1139. In P. H. A Sneath, N. S. Mair, M. E. Sharpe, and 

### J. G. Holt (ed.) Bergey's manual ofsystematic bacteriology.. Williams and Wilkins (Baltimore). . Teague, W; Martin and PhillipJ. Brumm. 1992. Commercial enzymes for starch hydrolysis products, pp.46-77. In 

### F. W. Schenck and R E. Hebeda (eds.) Starch HydrolysisProducts, VCH · Publishers, Inc., New York, NY. . . .' ,

· B. amvloliauefaciens exemption 5**~.'** III 

## .- 

# 'E ·0



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.

### ~.


### . ·"Z 

# I .. '.**1~**

/, 0**K--~'**



### , , Welker, N. E. and 

### L. 

### L. Campbell. 1967.

### Unr~latedness ofBaci;lus amyloliquefaciens and Bacillus subtilis. 

### J: Bacterial. 94:1125-1130. , '.
.

B. amyloZiqueJaciens exemption 6 .!It! III '. ,-' '''0

Ii. 

# o Z· 

# g o 

# z 

# • j'ffl - - i -Enzyme Business Toxicology To: PHva Copy: ABe' From:' ASB 27th October 1997 ED-9714960 ASBfDSc 

# .... 1/')' --.'!I""'"- "0 'L. 

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# .~•.

.Re : Evall1ation of

### the**pathoge~ic** potential ofBacillus amyloliquefaciens.. INTRODUCTION ,·f,

.

### T6investig~tethe potential

### ~fBaciUusamyloliquefaciensto cause disease in huinansalite"'

rature survey was initiated. The search was designed 10 cover case'stories of infection and .' intoxication saused by the misroorganism:··· .

..\ .EVALl,JATION' 

### OF THEPATHOGENIC POTENTIAL . '. References on ca!3e stories describing

### dis~a~e caused by 

### /? amyloliqiJefacienswas . searched forin**medica~** and microbiological handbooks and through a literature search in · relevant data bases, In medica!

### andmi~rol:Jiological handboQks B. arT/ylofiquefaciens iseith'erdescribed as non • - f" • -, - , '. " " " ' • • I . . . pathogellic or. notmentioned (ref. 1, 4; 5 and 6). • . ....... . • .., . . . '. . . . The

### h;lfldb~okof BG

### Chem~e 1992classifies

### B.~mylofiquefacilins as not.constituting a risk, · .to humci'nsor vertebrates (group 1) (ref. 2). ' . ..... . . .'. .' ..... '.

The search

### wasGar~iedoutin thefollowirig databases: MEDLINE

### (1985~1997), CAB (1973- .... 1997), TOXLINE**(1965~**1997),EMBASE (1974-1997), FOOD SCIENCE:.& TECH. ABS. . (1969-1997),PASC(1984-1997) andFOODLlNE:Food Sci & Tech (1972-1997), and the · ·following keywords were lJsed: TOXicity, pathogenicity or pathogen, intoxication andpois'on-

ing. . . . The search in databases resulted

### insixtee~ references on B. amyJoJiquefaciens. None of these described case stories of human infection or intoxication caused by B. amyJoJiquefaci- ·ens. CONCLUSION The search in medical handbooks and relevant data bases sh.ow that B. amyJoJiquefaciens is to be considered as non-pathogenic to humans and anirnals. The result ofthis survey sup- . Darts thp. -r.nnr.1t**l~inn** rl r::nAln in nl**Ihlir'~ti,......n** rli.",-.., fC'c-i".-. thn C'....,f",t" ""'-.f.D ........... , ,{r.f;..... , , ....**~** .........**:~ ~** **~ I_~C** 

### REFERENCES 1.· BalowsA, Hausler W.J., Herrmann K.L., Isenberg H.D., ShadomyH.J. 1991. Manual of Clinical Microbiology, 5th edition. American Society of Microbiology, Wasington D.C.

. . .. .. 2. BG Chemie. 1992. Sichere Biotechnologie. Eingruppierung biologisher Agenzien: Bake terien: BG Chemie Merkbfatt B 006 1/92. Jedermann-Verlag Dr· Otto Pfeffer oHG, ·Hei" delberg. 3. de BOer A.S. and 8; Diderichsen1991.0nthe safety of Bacillus suhtilisand Bacillus amyloliquefaciens : a review. Applied microbiology and biotechnofogy36: 1-4.. ·4. .Gorbach S.L., Bartlett

### J,G.,Bla~krow N.R. 1992. infeciious Dise.ases. W.B. S"<lunders Company, Loridon. .. ... 5. . Howard B.J.,

### Kl~as II

### J~. V\leissfeJclAS., Rubin S.J.., Tilton RC.19S7. Clinical and Pathogenic Microbiology. The C.V. Mosby Company, ,!oronto. 6.. Krieg N.R.,·HDltJ.G, 1984:.Bergey's Manual of Systemic Bacteriology. W1lHams& Wilkins, Bclltimore. . .r

) ."

·i