UNITED STATES ENVIRONMENTAL PROTECTION AGENCY WASHINGTON, D.C. 20460 Office of Chemical Safety and Pollution Prevention 

### MEMORANDUM June 06,2014 

### SUBJECT: Evaluation of new serum testing and field residue decline st udy for BLAD



### FROM: EPA Reg. #: 84876-R Chemical C lass: Biochemical PC Code : 030006 Miachel Rexrode, PhD, Senior Biologist I S / 06 1 06 12 014 Biochemical Pesticides Branch Biope s ticides & Pollution Preventio n Division (7511P) 

### TO: Menyon Adams , Regulatory Action Leader / S/ 06/06/2 014 Biochemical Pesticides Branch Biopesticides & Pollution Prevention Division (7511 P)


### ACTION REQUESTED The applicant, Ceres Internatio nal LLC (CEV), has generated and submitted additional information regarding BLAD that includes field residue decline studies and sera testing. The end-use product, PROBLAD PLUS (88306-E), is a n ew broad spectrum biofungicide that is be used on various crop s and ornamentals. The product was registered on February 2013with a tolerance exemption (78 FR 17600-17604 ; March 22, 20 13). The US Food and Drug Admi ni st ration raised concern regarding BLAD 's potential for allergenicity to populations that are sens itive to lupin s and peanuts since BLAD i s associated with the allergenic protein, Bconglutin that is found in leg ume plants like lupin s. After meeting with CEV regarding this concern the Agency suspended the registra tion and tolerance exemption until new information could be generated to sa ti sfy the se issues. The recent submission of new/enhanced BLAD sera test and field residue decline studies have been reviewed by the Agency as follows:Residue Testin g (MRID 49319401) T he applican t, CEV, previously submitted field residue studies on grapes, s trawberries and to ma toes using th e ma x imum PROBL A D PL US a pplication rate of0.75 lbs ai/A (five broadca st fol iar applicati ons). The s tudies show that there were no quantifiable resid ue s (LOQ = 0.0 2 ppm) of BL A D to tomatoes ( < 0.0067 ppm) after the l ast ap plication and all residues on grapes and strawberries were at o r below the Limi t of Detection (LOD 0.005 ppm) for a ll samp l es (M RID 493 1 9401).

T he Agency also requested that CEV conduct field trials u sing BLAD at exaggerated levels (5x 1 Ox) in order to d etermine the rate of BLAD residue degradation. Th e app li cant con duc ted these fie ld trials using 5X because the 1 OX co n centratio n would ha ve b ee n phytotoxic. CEV also used th e residue measurements from th e exi st in g field stu dy to calculate a decline rate ( half-life) , e.g. th eo retical anticipated resid ue s over tim e in straw berries and tomatoes u si n g the 5X trial s (3.75 lbs AVA) after 5 s u ccessive applications (Figure 1 ). The decl ine curves for b oth crops show a similar pattern with a h alf-life of2 days. 35 30 - .a **~** 25 - c: 0 +l 20 - "' .. ... c: Cll 15 u c: 0 u "0 10 "' iii 5 0 0

Fig ure 1- Declin e Ra te. afte r the l ast of 5 wee k-apart co nsecut ive applications . all at 5 times ma ximum r ate (3. 75 lb ai/A l 1

I I L---t-- -l--- 1

**~;~ Tomato**  Straw berry **--~-** 2 3 4 5 6 DALA 7 The Agency u sed this information to calculate a theo r et ical rat e of degradation (0 .4215) of BLAD using a first orde r degradation mode l (Appendix A.O).Serum Testing (MRID 49276602) Co dex (2003, Annex III) provided guideline s for assessing protein allergen icity and if a concern i s raised additional testin g is required. The bioinformatic s analys i s for BLAD identi fied simi larities to lupin and peanut prot e ins thu s justifying the need for a sera test of a sen s itive population (allergic response to lup in and/or peanuts). CEV conducted Skin Prick Test (SPT) in order to establis h a sampl ing population that was sensi ti ve to lupins and/or peanuts. Sera from these patients was then tested for a ll e rge nic reaction to lupins/peanuts (control ) and to BLAD but there were uncertainties about the results from some of the corresponding immunob lots . The Agency expresse d concern over the veri fication of res ults tha t were pre sente d in the original document and s ug gested that thi s may b e a reflection of the poor resolution of the gel photos that were s ubmi tte d. After reque sti n g a nd receiving larger gel photos the Agency sti ll had concerns rega rdin g that the expression ofpositive contro l s in several gels (i.e. 2, 3, 1 1 , 1 6 , 17 , 22 , 23, and 2 4). CEV agreed that o nl y faint signals were observed for these positive contro l s and decided to redo the test using a m ore concent rated so lution of the test serum . In the ori ginal test a 1/20 dilution of the patient sera was used but the current tests were up g raded wit h a 1110 dilution thus producing a stronger s ignal. CEV also added 5 new patient sera in addition to incre as in g the sera concentration of the 8 previous sera of concern. The re s ul ts of the second s tudy show that a p os itive contro l signal was produc ed in the immunoblots corresponding to the individual sera, and no signal was produced for BLAD with all tested sera. The selected sera fro m individua l s sensiti ve to lupin and/o r peanuts did not show reactivity to BLAD , suggesting that BLAD appears not be a potential allergen to this sensit ive population (Figure 1.0 and 2.0). Co nclusions T he applicant, CEV , h as submi tted field residue studies on grapes, s trawberries and tomatoes using the maximum PROBLAD PLUS appli cation rate of 0 .75 lbs ai/A (five broadcast foliar applications). The stu dies show that there were no quantifi ab le res idues (LOQ = 0.02 ppm) of B LAD to tomato es (< 0.0067 ppm) after the last application and all residue s on gra pes and strawberries were at or below the Limit of D etection (LOD 0.005 ppm) for a ll samples (MRID 493 1 940 1 ). The Agency used the information regarding the decline rate of BLAD and calculated a rate of degradation (0.42 15) assuming first o rd e r degradation (Appendix A.O). The sera test representing a sensiti ze d popul ation to lup ins and/or peanuts s howed positive reaction to lupins and/or peanuts but no r eaction was no ted on the immun ob lot to BLAD. Thi s suggests that B LAD appears to not be a potential allergen. Negat ive resu lts from this serum testin g (dose response) of25 patients (well defined population) wo uld show t hat BLAD appears to not be an allergen. Therefore when combi ned wi th field magnitude of residue data demonstrating rapid decline ofB LAD following application, th e negati ve in vitro immunologicalstudy could b e used to su pport th e existing tolerance exemption (at the highest le vel that BLAD was tested). Appendix A .O Calculation of BLAD Degradation RateAssuming a 

### Y2 life of 2 hours a rate of degradation was determined as follows : I n [A ] 

### = -k(t) +In [Ao] I n (0.012 ppm) = -k(2) + In (0.279 ppm)

- 4.422 

### = -2k + (- 3 . 579) 2k = 4.422 - 3.579 2k = 0.8438 k 

### = 0.4215 rate of degradation This rate of degradation was applied to the initial nominal concentration and extrapolated for the duration of the various test periods . The example in Table 1 . 0 shows the assumed concentration of active ingredient over a 6 hour period. 

### Table 1.0 Approximate Concentrations of BLAD Relative to First Order Degradation and 

### T" "tb D d . R t C I I t d t 0 4215

1mew1 a egra ahon ae a cu a e a 

# . Concentrations**{pJ~m)** Initial lhr 2hrs 3hrs 4 hrs 5hrs 6hrs Concentration 0.0279 0.0287 0 .012 0.0095 0.0052 0 .003 0.002 These values correspond to actual residues that the applicant found for strawberries (0.0246 t o 0.005 ppm after 5 hrs).



### Appendix B . OImmunoblot analysis for BLADThe reactivity to BLAD was assessed using the sera from a sensitive population (lupin and/or peanuts). Figure 1.0 represents the electro - transferred nitrocellulose membrane stained for total

protein with Ponceau-S and shows the polypeptide pattern of lupin , P - cong lu tin, BLAD and peanuts. Figure 2.0 shows the reaction of sera from the sensit i ve population to the a lle rgens (positive control) and to BLAD. There appears to be no response to BLAD for this sensitive population. Figure 1.0 Total protein stained with Ponceau-S. a) Lupin; b) P-conglutin; c) BLAD; d) Peanut tota l protein and MW- molecular marker MW a b c d Figure 2.0 Reactivity analysis of: (a) Lupin seed tota l protein; (b) P-conglutin from Lupin seed; (c) BLAD polypeptide from PROBLAD PLUS fungicide ; (d) Peanut total protein, probed withserum from patient (2); (3); ( 16) ; ( 17) ; (23); (2 6); (2 7 ); (28 ); (29) and (30). T he arrow sho ws th e p os ition of the polyp e ptide BLAD.

b



### P atient serum 2 -High concentration Patient serum 3 -High concentration

Patient serum 3 -High concentrationc Patient serum 16 - High concentration b Patient serum 17 - High concentration

### Patient serum 23 - High concentration MW 

### a b c d



### Patient serum 26MW a b c d Patient serum 27 MW a b c d 25k.Oa Patient serum 28MW a b 

### c d .



### Patient serum 29

MW a b c

2Sk0.


### Patient serum 30