### UNITED ST ATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON , D.C. 20460



## JU L 0 3 

## 2 0

14 OFF ICE OF CHEM ICAL SAFETY AND POLLUTION PREVENTION


### MEMORANDUM
SUBJECT: Review of the Registrant ' s r es p o n se t o data deficiencies fo und b y th e Age nc y in its review of Product C he mi str y , and Toxicity Studies fo r the reg i s trati o n of two products: NoF l y Tec hni ca l and NoF l y WP co nt aini n g the new A I 

### Isa r iafumosoroseus s tra in FE 990 1
. 

### FROM : Ib rahim S. Barsoum , Ph.D. , Microbiologist,

# 11~~~ b / 25 / 

# l ) l Microbial P est ic id es Branch , 

### THRO UGH : Biopesticides and Pollution Prevention Division**~** John L. Kough , Ph.D
. , Senior

# Scien**ti s~**  v



### TO: Microbial P es ti c id es Branch , Biopesticides and Pollution Prevent ion ision A Jeannine Kausch, Regulatory Ac ti on Leader Microbial P es ticid es Branch , Biopesticides an d Pollution Preventio n Division (75 11 C)


### ACTION REQU ES TED: To review the Registrant ' s res p o n se (letters d a te d 7 / 22/ 20 1 3 & 5 / 1 6/ 20 14 ) to th e de fici e nci es fo und b y the Agency in its review of P rod uct C he mi s try , and Toxic ity St

udi es oftwo products : NoFly Tec hni ca l (E P A Reg. no. 733 14

- 7) a nd NoFly WP (EPA Reg. no. 73314-6) containing th e new A I 

### Jsariafumosoro s eus strain FE 990 1.


### CONCLUSION: ACCEPTABLE T he regis trant h as s u ccessfu ll y r espo nd e d to the fo ll owi ng deficiencies: 1
. Demonstrate that th e re i s no sig nificant tox i c metabolites produc ed by the TGA I. 2. C han ge the gen u s n ame of the active i n gre di e nt fr o m 

### Paecilomyc e s to 

### I saria
. 3. Submit Storage Stability & Corrosion C h arac t eri st ics st udi es of the EP. 

### C O NTA I NS FIFRA CONFIDENTIAL B US INESS INFORMATION

### DATA REVIEW RECORD Active In g re di e nt: Product Na me: Co mpan y Name: Reg. No: C he mi ca l N umb e r: Deci s i o n N umb e r: DP Barcode: MRJD No: 

### BACKGROUND: l saria fu m oso ros e us s train FE 9901 NoF ly Tec hni cal, a nd No Fl y Wettabl e P owder Novozy m es Bi oAg , Inc . , Bro o kfield , WI 733 14-6 7 333 14

- 7 11 5003 481554 , 48 1 555 , 481 557 41761 2 , 42040 1 , 4 204 06 49118301 , 49378401



### l sa ri afim10soroseus i s a n aturall y -o ccu rrin g fungus in m ost countries of the wo rl d and occ ur s in vario u s so il types at ve ry low de n s iti es. It s t a rge t organi s m s are th e w hitefl i es. In it s n at ura l e n v i ronme n t , I. 

### fumosoroseus stra in FE 990 I i s di s p e r se d as conidia o r b lastospores , e ither by air o r wate r m oveme nt or by o th er in sec t s an d/ o r mites. In an earli er s tudy (MR ID No. 4829 1 2

- 0 1) , extracG of biom ass a n d culture filtrate d e ri ved fro m s ti ll cu ltu re and st irred tank reac tor (ST R ) fe rm e ntati o n s of 

### J saria fumosoroseus s train FE 990 I were a nal yzed to detect the presence of seco nd a ry m e tabo lit es and to eva lu a te th e ir cytotoxic ity on hum a n lun g cance r ( H e p- 2) and ca nin e k idn ey (MOCK) ce ll lin es. The a n a lys i s b y HPL C combined w ith c hro m atog r ap h y revea le d th e prese nce of beauv e rolid es Ma nd I in th e ex tract s from s till c ultures w ith n o s ig nifi ca nt leve l s of the se compo und s d e tecte d in th e STR extracts. T h e g rowt h inhibiti on assay o n ce ll lin es re vea le d a dose depe nd e nt cyto to x ic ity in th e extrac t s from s till cul ture that i s 6

- 

### ! OX hi g h e r t ha n in st i rred c ul t ures , a nd no a ntiproli fera ti ve act i v ity was detected in a n y of the extrac ts. The auth o r s deduced fro m th ese s tudi es th a t fe rm e nt at i o n of 

### Jsariajitmosoroseus stra in FE 990 I in s tirre d tank reac t o r s d oes n ot produce u nin tentional compound of tox ic concern. The Age n cy , however, concl ud e d th a t th ere i s a potentia l for the presence of tox ic metabolites in th e techn i ca l produc t , even t h ough a t a lowe r leve l than in s till cultures ; and as s uch , th e Age nc y cannot s upp o 1 1 u se of th e product o n food crops. To pro ve th e abse n ce of toxic metabolites , the reg i s tra nt did fi ve bat c he s ana lys i s o n the techn ica l product , manu fac tured at th e actual product io n s ite, and demon strate d t h at th e re are no s ig nifi cant toxic m etabol ites produced. A l so , in th e origina l data s ubmi tted, a viab l e co unt t es t (CFU / g) of th e fo 1 mu latio n s h owed a 10

- fo ld re ducti o n ove r th e s tora ge p eri od of s i x m o nt h s se t a t 4°C ( ± 2 °C). There was n o sig nificant differe n ce in th e p h ys ica l tes t res ult s b efo re and after s t orage. H oweve r , s in ce th e o n e batch of E P t h at was tested was no t produc e d a t th e prop ose d manufacturin g s ite , t h e Age ncy h as as k e d that th e reg i strant s ubmit a s tud y of sto rage stab ility a nd corrosion char ac te ri s ti cs of th e E P fo r s ix months at room te mp erat ure a nd a t 4 °C. 

### Regi s trant 's Respons e to the Deficiencies found by the Agencv in its r ev iew of product chem i s try, and ac ute toxicity studi es of NoF lv Tec hnical and NoFly WP conta ining the new AI 

### Isa ria {umosoroseus stra in FE 9901

2

### Deficiency 1: Discussion of formation of unintentional ingredients. The Agency is concerned that 

### ! sar ia fumosoroseus strain FE 9901 is capab le of producing toxic metabolites in fermented cultures used in the manufacture of No Fly Technical and consequently cannot issue an exemption from the requirement of a tolerance for re s idu es of 

### Isaria fumosoroseus strain FE 9901 for food u se. 

### Registrant's Response: (M RID # 49 11 8301) Five batches ofNoFLYTM Technical, containing 

### !sariafumo soroseus strain FE 9901 , were eva lu ated for the presence of beauverolides , beauvericin, and 2, 6 -p yri dinedicarboxylic acid. Each of the metabolites was quantified in the pg/ g and low μ gig ranges. In this study , the most prevalent beauverolide peptide , beauverolide I , was identified a l ong with 14 others in the class according to their chromatographic and mass spectral properties. Beauverolide I was determined to be pre se nt at a range of (Ta ble 1 ). Table 1. Beauverolide Metabolite Formation No Fly Technical BVD-1 Standard Coeff

. Of

Sample (μg/g) Deviation (±) Variation(%)

Beauverolide I was the predominant b eau vero lide peptide d e tecte d among s t 15 beauverolide peptide species. Beauverolide-1 was approximate ly of the overall p ep tides combined, and 100 % of the peptide s combined are well below 0
. 1% of the NoFLYTM Technical TGAI ; therefore , no metabolite formation rises to a level of toxicological**concern ~**  '-

### CONCLUSION: ACCEPTABLE Deficiency 2: Change the genus name of the active ingredient from Paecilomyces 

### to Isaria
. The name of the TGAI needs to be updated on the data matrix to reflect the current taxonom y. Also, the new CSF and label should reflect the most common taxonomy i .e. th e genus name should b e changed from 

### Paecilomyc es to 

### /saria
. 

### Registrant's Response: The registrant ha s submitted revised data matrix , CSF and label with the genus name changed from 

### Paecilom yces to 

### Jsaria. 

### CONCLUSION: ACCEPTABLE

3

### Deficiency 3 : Submit Storage Stabilitv & 

### Corrosion Characteristics studies of the EP A s torage stabili ty s tud y mu st b e c ondu c te d u s in g th e prop ose d fo rmul a tion ofNoF l y W P fr o m th e p rop o se d m a nu fac turin g s ite. 

### Registrant' s Response: (M RID # 4 93 7 84 0 1 ) A n initi a l diluti o n i s pe rfo rmed by d i lutin g 0
. 1 g of t ec hni ca l grad e NoF l y into 10 0 ml s te ril e ph os ph at e bu ffe r o r s te ril e wat e r. T he fl as k co nte nt s are sti r -mi xe d 3 00 rpm fo r 15 minut es in o rd e r t o in s ure effec ti ve s p ore di s p ersa l. A 1: 1 0 diluti o n i s p erform e d. A 1 0 μ l aliqu o t from thi s diluti o n bl a n k i s tra n sferre d to a co untin g c h a mb e r ( h ae m ocyto mete r , imp roved Ne ub au er, Bri g ht-lin e TM , Bu ffa lo , NY). Eac h diluti o n i s exa min e d at 400 X ( tota l m ag nifi ca ti o n ) w ith a li g ht mi c ro sco p e. Diluti o n leve l s are a dju s te d if necessa r y t o ass ure th at th e numb er of s p o res p e r fi e ld is b etwee n 50 and 200. A t o ta l of I 0 s qu a res a re co un ted a nd th e m ea n numb e r o f s p o res co unte d i s ca lcul a te d. Th e t o t a l numb er of s p o res p e r g r a m is ca lc ul a te d as fo ll ows: N = [M x (2.5 x 10 5 ) x d i luti o n fac t o r x rec i proca l g/ ml ( m ass/ m l in o ri g ina l fl as k) ] / numb er of s qu a r es c o unte d
. M = to ta l numb er of ce ll s in a ll s qu a res co unte d (a s qu ar e i s co mpri se d of 1 6 g rid s) 2
. 5 x I 0 5 i s th e rec ip roca l vo lum e in ml of each s qu a re Table 2

. V i able spores (CFU/g) in N oFl y W P up to 6 months a t room t em p e r at u re a n d 8 ° C
1 mont h 1 mo n t h 3 m ont h 3 m onth 6 mo nth 6 mont h L ot T=O @RT @BC @R T @BC @RT @BC
538 1.4 x lO lU 5

. 4 x 10 " 9. 1 x 10 " 1.2 x lO lU 1

. 7 x lO lU 4 .3 x**10 ~**  1.4 X lQ l U 540 7.7 x 10 " 3

. B x 10 " 1.1 x lO lU 7 .3x 10 " 1 .6 x lO lU 0 1.0 x lO lU

T he d at a s h ows th at No Fl y WP (7 33 14- 6) m a inta in s th e lower ac t iv it y tite r of 2 x 1 09
, as n o t e d o n th e a pp rove d CSF, fo r 3 m o nth s at roo m t e mp era ture and 6 m o nth s at 8°C. B ase d o n th e ab ove res u lt s, the prop ose d wo rdin g o n th e NoF l y W P (7 33 14- 6) l ab e l in th e Sto rage and Di s p osa l sec ti o n co nce rnin g s ta bilit y i s as fo ll ows : " Sto re fo r 6 m o nth s at 4 6 ° F ( 8 °C). S t o ra ge at roo m t e mp era ture (76°F, 23°C) w ill re du ce th e s he lf li fe to 3 m o nth s. " Produ c t was s t o re d in co mmerci a l p ac kag in g for th e durati o n of the stud y a nd w a s monitore d fo r co rr os io n o r o th er d amage thr o u g h o ut th e s tud y b y v i s u a l i n s p ecl io n . No co r ros io n was n o ted d urin g th e du rati o n of t he st ud y . 

### CO N CL U SIO N: ACC E PTABLE

4