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 study for BLAD 

FROM: 

EPA Reg. #: 84876-R 

Chemical Class: Biochemical 

PC Code : 030006 

Miachel Rexrode, PhD, Senior Biologist IS/ 0610612014 

Biochemical Pesticides Branch 

Biopesticides & Pollution Prevention Division (7511P) 

TO: Menyon Adams, Regulatory Action Leader /S/ 06/06/2014 

Biochemical Pesticides Branch 

Biopesticides & Pollution Prevention Division (7 511 P) 

ACTION REQUESTED 

The applicant, Ceres International 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 new broad spectrum biofungicide that is be 

used on various crops 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 

Administration raised concern regarding BLAD's potential for allergenicity to populations that 

are sensitive to lupins and peanuts since BLAD is associated with the allergenic protein, Bconglutin that is found in legume plants like lupins. After meeting with CEV regarding this 

concern the Agency suspended the registration and tolerance exemption until new information 

could be generated to satisfy these issues. 

The recent submission of new/enhanced BLAD sera test and field residue decline studies have 

been reviewed by the Agency as follows: 
Residue Testing (MRID 49319401) 

The applicant, CEV, previously submitted field residue studies on grapes, strawberries and 

tomatoes using the maximum PROBLAD PLUS application rate of0.75 lbs ai/A (five broadcast 

foliar applications). The studies show that there were no quantifiable residues (LOQ = 0.02 ppm) 

ofBLAD to tomatoes (<0.0067 ppm) after the last application and all residues on grapes and 

strawberries were at or below the Limit of Detection (LOD 0.005 ppm) for all samples (MRID 

493 19401). 

The Agency also requested that CEV conduct field trials using BLAD at exaggerated levels (5x 

1 Ox) in order to determine the rate of BLAD residue degradation. The applicant conducted these 

field trials using 5X because the 1 OX concentration would have been phytotoxic. CEV also used 

the residue measurements from the existing field study to calculate a decline rate (half-life), e.g. 

theoretical anticipated residues over time in strawberries and tomatoes using the 5X trials (3.75 

lbs AVA) after 5 successive applications (Figure 1). The decline curves for both crops show a 

similar pattern with a half-life of2 days. 

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Figure 1- Decline Rate. after the last of 5 week-apart consecutive applications. 

all at 5 times maximum rate (3. 75 lb ai/Al 

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The Agency used this information to calculate a theoretical rate of degradation (0.4215) of 

BLAD using a first order degradation model (Appendix A.O). 
Serum Testing (MRID 49276602) 

Codex (2003, Annex III) provided guidelines for assessing protein allergenicity and if a concern 

is raised additional testing is required. The bioinformatics analysis for BLAD identified 

similarities to lupin and peanut proteins thus justifying the need for a sera test of a sensitive 

population (allergic response to lupin and/or peanuts). 

CEV conducted Skin Prick Test (SPT) in order to establish a sampling population that was 

sensitive to lupins and/or peanuts. Sera from these patients was then tested for allergenic reaction 

to lupins/peanuts (control) and to BLAD but there were uncertainties about the results from some 

of the corresponding immunoblots. The Agency expressed concern over the verification of 

results that were presented in the original document and suggested that this may be a reflection 

of the poor resolution of the gel photos that were submitted. After requesting and receiving larger 

gel photos the Agency still had concerns regarding that the expression of positive controls in 

several gels (i.e. 2, 3, 11, 16, 17, 22, 23, and 24). CEV agreed that only faint signals were 

observed for these positive controls and decided to redo the test using a more concentrated 

solution of the test serum. In the original test a 1/20 dilution of the patient sera was used but the 

current tests were upgraded with a 1110 dilution thus producing a stronger signal. CEV also 

added 5 new patient sera in addition to increasing the sera concentration of the 8 previous sera of 

concern. 

The results of the second study show that a positive control signal was produced in the 

immunoblots corresponding to the individual sera, and no signal was produced for BLAD with 

all tested sera. The selected sera from individuals sensitive to lupin and/or peanuts did not show 

reactivity to BLAD, suggesting that BLAD appears not be a potential allergen to this sensitive 

population (Figure 1.0 and 2.0). 

Conclusions 

The applicant, CEV, has submitted field residue studies on grapes, strawberries and tomatoes 

using the maximum PROBLAD PLUS application rate of 0.75 lbs ai/A (five broadcast foliar 

applications). The studies show that there were no quantifiable residues (LOQ = 0.02 ppm) of 

BLAD to tomatoes ( <0.0067 ppm) after the last application and all residues on grapes and 

strawberries were at or below the Limit of Detection (LOD 0.005 ppm) for all samples (MRID 

493 19401). The Agency used the information regarding the decline rate of BLAD and calculated 

a rate of degradation (0.42 15) assuming first order degradation (Appendix A.O). 

The sera test representing a sensitized population to lupins and/or peanuts showed positive 

reaction to lupins and/or peanuts but no reaction was noted on the immunoblot to BLAD. This 

suggests that BLAD appears to not be a potential allergen. Negative results from this serum 

testing (dose response) of25 patients (well defined population) would show that BLAD appears 

to not be an allergen. Therefore when combined with field magnitude of residue data 

demonstrating rapid decline ofBLAD following application, the negative in vitro immunological 
study could be used to support the existing tolerance exemption (at the highest level that BLAD 

was tested). 

Appendix A.O Calculation of BLAD Degradation Rate 
Assuming a Y2 life of 2 hours a rate of degradation was determined as follows: 

In [A] = -k(t) +In [Ao] 

In (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" 1mew1 "tb a D d egra ahon . R ae t C a I cu I a e a t d t 0 . 4215 

Concentrations {pJ~m) 

Initial lhr 2hrs 3hrs 4hrs 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 to 

0.005 ppm after 5 hrs ). 

Appendix B.O Immunoblot analysis for BLAD 
The 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-conglutin, BLAD and 

peanuts. Figure 2.0 shows the reaction of sera from the sensitive population to the allergens 

(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 

total protein and MW- molecular marker 

MW a b c d 

Figure 2.0 Reactivity analysis of: (a) Lupin seed total protein; (b) P-conglutin from Lupin seed; 

(c) BLAD polypeptide from PROBLAD PLUS fungicide; (d) Peanut total protein, probed with 
serum from patient (2); (3); (16); (17); (23); (26); (27); (28); (29) and (30). The arrow shows the 

position of the polypeptide BLAD. 

b 

Patient serum 2 -High concentration Patient serum 3 -High concentration 

Patient serum 3 -High concentration 
c 

Patient serum 16 - High concentration 

b 

Patient serum 17 - High concentration 
Patient serum 23 - High concentration 

MW a b c d 

Patient serum 26 
MW a b c d 

Patient serum 27 

MW a b c d 

25k.Oa 

Patient serum 28 
MW a b c d. 

Patient serum 29 

MW a b c 

2Sk0. 

Patient serum 30