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7Noveinbt?r, 1997 . NOV 1 3 1997 

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. Chief, New Chemicals Branch 

Chemical Control Division . 

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}~ Office of Pollution Preventlon and.Toxics 

U.S. Environmental Protection Agency' 

401MS(SW . 

Washingto[1, DC 20460 . 

.. Re:' . letter of Application .... .' . .'. . ..... '.. '. 

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

orgarJism be included.in§.725.420 as a candidate recipient 

microorganism for tiered¢xemplion' . . 

. On Deoember 30, 1994,. Novo Nordisk BibChemNorth America,lno. 

(Novo Nordisk) submitted to EPA a fetter requesting oonfirmation .that 

the designation ofd:3aoillus subtilis, as afeoipient organism eligibl~ for 

. tiered eXemption. included· Bacillus amyloliquefaciens. 

I ri the EPA's Final regulation Under .the Toxic substances Control Act: . 

. ' ASumma,yof the 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 of Application 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 Nordisk BioChem 

North' Amerlca~ I~c. 

state Road 1003 

Box 576 

·fra~klinton. NC 27525-0576 

Tel. 919-4943000. 

FAX·919-4943450 

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inthEfliterature and reference manual~. Most of the commercially-. 0 . . ... > · available intermediate temperature stability alpha 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, in essence, 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 requirements Jar a tetter of Application itemized in§ 725.67 list . 

several 'general provisions that should be provided to· Show that the 

requested exemption will not present an unreasonable risk of injury to 

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 2 
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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 telephone at 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) 

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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 tests fail 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 

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In a nLimerical classification study using 118 characteristics of 3680 

species of· Bacillus, the· species B. thuringiensis and B. .cereus 2 

clustered together 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 authors stated 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 of safe ,commercial use for the microorganism 

.. Baci[fus amyloliquefaciensisone of the 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 of thenomenCiafure . 

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· (T eagueand " -,' • • ..... - - - - '- < -' 

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 

made from 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 subject to TSCA , . 

· TSCA uses of amylases include desizing of,textiles and starch 

· modification for sizing of paper and industrial ethanol. TSCA uses of 

proteases include' cleaning aids in det~rgents anddehairing and 

bating inthe leather industry (Erikspn, 1976). 

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E) Studies which indicate potential . for the microorganism to 

cause adverse effe~tsto health or the environment 

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, 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 of the 

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 the species 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<;; pyprnntinn 
REFERENCES, 

7 CFR 330, ef seg., as amended. 

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'Alexander, M. ,,1977. Introduction to Soil Microbiology: John Wiley 

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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 and Wilkins Co., 'Baltimore; M~ , ' ' 

De Boer, A. S. and B. Diderichsen. ,1991. ,On the safety of BacillUs 

SUJjfi/iS8r)d Bacillus amyloliquefadens: A review. App1. Microbial: 

Biotech. 36:1-4. 

'Erikson, RJ:1976, "Industrii::d applications of the bacilH:,A review 

and prospect!Js,pp. 406-419. in p. Schlesinger (ed), Microbiology. 

,'AmericanSociely for Microbiology, Washington~ DC. 

Fukutomo, J. 1943.' Studies on the production ofbaC\erial amylase. '. 

I. ISolation of bacteria' 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 of Agriculture, Washington, DC, 

Harwood, Colin R.19,92. Bacillus subtilis and its relatives:, 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 the genus 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; 

· 

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'Isolation; arid Identiticationof 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.) 

The Aerobic 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 of systematic 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 

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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 

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Enzyme Business Toxicology 

To: PHva 

Copy: ABe' 

From:' ASB 

27th October 1997 

ED-9714960 

ASBfDSc 

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.Re : Evall1ation of the pathoge~ic potential of Bacillus 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:··· . 

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.EVALl,JATION' OF THE PATHOGENIC 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 is eith'erdescribed as non • - f" • -, - , '. " " " ' • • I . . . 

pathogellic or. not mentioned (ref. 1, 4; 5 and 6). • . ....... . • .., . . . '. . . . 

The h;lfldb~okof BG Chem~e 1992 classifies 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 of this 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; Diderichsen1991.0n the safety of Bacillus suhtilis and 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. . . 
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