Document ID: EPA-HQ-OPP-2010-0081-0005
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2011-03-18T04:00Z

MEMORANDUM					

SUBJECT:	Review of Toxicology Data for Metarhizium anisopliae F52 in
Support of a Section Three Registration for Metarhizium anisopliae F52
from Taensa, Inc., Fairfield, CT.

FROM: 	Chris A. Wozniak, Ph.D., Primary Reviewer

Microbial Pesticides Branch				

Biopesticides and Pollution Prevention Division	

THROUGH: John L. Kough, Ph.D., Senior Scientist

Microbial Pesticides Branch

Biopesticides and Pollution Prevention Division

TO: 		Leonard Cole, Regulatory Action Leader

Biopesticides and Pollution Prevention Division

ACTION REQUESTED: 

Taensa Inc. has applied for a Section 3 registration for indoor and
greenhouse use of Metarhizium anisopliae strain F52. This fungus in an
entomopathogen and will be used to control household and greenhouse
insect pests. This is considered a non-food use and therefore no
associated tolerance is requested at this time. The attached data
evaluation reports were originally reviewed by Agency contract employees
and were subsequently reviewed by BPPD scientists.

BACKGROUND:

The active ingredient, M. anisopliae Strain F52, will be produced using
two different manufacturing processes in place at four different
locations (manufacturers). M. anisopliae F52 is proposed for use in
greenhouses and indoors on non-food applications. This deuteromycetous
fungus has a host range primarily affecting coleopterans of the families
Elateridae and Curculionidae, although other insect groups are known to
be within the host range of this pathogen. This species is known
worldwide from insects and non-insect sites, such as soil, river
sediments, associated with nematodes, and as a saprophyte on organic
detritus.	



DATA REVIEW RECORD

Active Ingredients:	Metarhizium anisopliae strain F52

Product Name:	TAE-001 Technical Bioinsecticide

ID No.:		072098-T TAE-001 Technical Bioinsecticide

Submission No.:	S569284

Chemical No.:	029056 Metarhizium anisopliae strain F52

DP Barcode:		D271484

Sponsor:		Taensa, Inc., 26 Sherman Court, P.O. Box 764, Fairfield, CT
06430

MRID/Study Titles:	448447-09 - Acute Oral Toxicity / Pathogenicity - Rat
(885.3050)

448447-10 - Acute Dermal Toxicity / Pathology Study of  Metarhizium
anisopliae Strain 52 in Rabbits		

448447-11 - Toxicity / Pathogenicity Testing of  Metarhizium anisopliae
Strain 52 Following Acute Intratracheal Challenge in Rats

448447-12 - Toxicity / Pathogenicity Testing of  Metarhizium anisopliae
Strain 52 Following Acute Intraperitoneal Challenge in Rats

448447-13 - Primary Eye Irritation Study of Metarhizium anisopliae,
Strain F52, in Rabbits

448447-15 - Dermal Sensitization Study of Metarhizium anisopliae, Strain
F52, in Guinea Pigs Using the Buehler Method

	

SUMMARY OF REVIEWS:

448447-09 - Acute Oral Toxicity / Pathogenicity - Rat (885.3050) -
Fifty-six males and 56 female CD Rats, approximately 58 days old, were
divided into treatment group (TG), killed treatment group (KTG) and
naive control (NC) groups. A shelf control (SC) group was also not
treated and housed with the TG group. TG group rats were dosed orally
with approximately 1.04 x 108 cfu of Metarhizium anisopliae per animal
in a 1 mL volume. KTG rats were treated with heat-killed test substance
and naive control group rats were not dosed.  Body weights were recorded
at the time of dosing and on days 3 and 7.  The test animals were
observed for clinical signs twice daily.  The animals were fasted
overnight prior to sacrifice for test material enumeration from tissue. 
Three rats/sex/group from the TG, KTG, and NC groups were scheduled to
be killed and necropsied on days 0, 3, 7, 14, and 21.  Due to the
clearance of the test material by day 3, the animals scheduled to be
sacrificed on days 14 and 21 were killed on day 7 and not necropsied.
The SC group rats were necropsied on day 7.  The blood, brain, lungs,
spleen, liver, kidneys, mesenteric lymph nodes, stomach and small
intestines, cecum, and feces were removed for microbial enumeration and
weight determination. Samples from these tissues and organs recovered
the test substance on day 0, but by day 3 the test substance was below
the detection limit.  Metarhizium anisopliae Strain F52 does not appear
to be toxic and/or pathogenic in rats, when dosed at 1.04 x 108
cfu/animal. 

Classification: Acceptable.

448447-10 - Acute Dermal Toxicity / Pathology Study of  Metarhizium
anisopliae Strain 52 in Rabbits - Five male and five female New Zealand
white rabbits were received from Kuiper Rabbit Ranch, Gary, IN.  The
male and female rabbits weighed 2.3-2.6 kg and 2.3-2.4 kg, respectively,
on the day of treatment.  The test material (2000 mg/kg body weight;
3.63-4.42 x 1010 cfu/animal) was applied evenly to the clipped dorsal
trunk in an area of approximately 10% (about 240 cm2).  The application
site was covered with a surgical dressing and a plastic film, then
secured with a lint-free cloth and an elastic adhesive bandage. The skin
was checked for irritation within one hour after patch removal and daily
thereafter for 14 days.   The rabbits were weighed prior to treatment
and on days 7 and 14.  The rabbits were euthanized on day 14 without
gross necropsy. No sign of overt toxicity was noted from any rabbit. 
Very slight to severe erythema was noted on all rabbits after patch
removal with clearance by the second week.  Very slight to moderate
edema was noted on one male and five females after patch removal with
clearance by day 6 of the study.  Eschar, scabs, cracking skin, and/or
scaly skin were noted on a few animals during the first week of the
study. All animals recovered from these irritation symptoms before  the
end of the study. No deaths and no clinical toxicity were observed.  The
acute lethal dose (LD50) is greater than 2000 mg/kg. 

Classification: Acceptable - Toxicity category III.

448447-11 - Toxicity / Pathogenicity Testing of  Metarhizium anisopliae
Strain 52 Following Acute Intratracheal Challenge in Rats - Eighty male
and 80 female CD®rats, approximately 70 days old, were assigned to
groups: treatment group (TG), killed treatment group (KTG), naive
control (NC), and shelf control (SC).  The TG group rats were dosed with
approximately 1.17 ( 108 cfu/0.1 mL/animal. Body weights were recorded
weekly (0, 7, 14, 21, 28, and 35). The test animals were observed for
clinical signs twice daily.  Five rats/sex/group from the TG, KTG, and
NC groups were killed and necropsied on days 0, 7, 21, and 35.  The SC
group rats were necropsied on day 35.  The blood, brain, lungs and
associated lymph nodes, spleen, liver, kidneys, and cecum were removed
for microbial enumeration and weight determination. Mean body weight of
males and females and mean body weight gains of males indicated no
statistically significant effects in any test groups during the study. 
The mean body weight gains of the females in the TG group were
statistically lower than the control groups between days 14 and 21. One
TG male (No. 515) and one NC female (No. 621) lost weight during the
first week. One KTG female (No. 605) and one NC female (No. 626) lost
weight during the second week. One TG female (No. 586) lost weight
during the third week.  One TG female (No. 590) lost weight during the
fourth week. No adverse signs were observed from any group. Three
females (Nos. 585, 586, and 589) in the TG group that were sacrificed on
day 21 showed brown and/or mottled lungs. All other rats had no gross
lesions. Decreased relative spleen, kidney, brain, and liver weights
appeared to be a transitory effect.  No statistically significant
effects in relative organ weights in other tissues were observed.  Since
these weight differences were transitory, the observation is considered
as not biologically meaningful and likely reflects the normal variation
present in organ weights of animals. Metarhizium anisopliae was only
detected in the rats treated with the test material (TG group).
Metarhizium anisopliae was detected in the lungs and associated lymph
nodes of males on days 0 and 7 and were below the detection limit by day
21. The test organism was detected in the lungs and associated lymph
nodes of females on days 0, 7, and 21 and were below the detection limit
by day 35. The test organism was detected in the cecum of males and was
below the detection limit by day 7. The test organism was detected in
the spleen of females and was below the detection limit by day 21.
Metarhizium anisopliae, Strain F52 does not appear to be toxic and/or
pathogenic in rats, when dosed intratracheally at approximately 1.17 (
108 cfu/animal.

Classification: Acceptable.

448447-12 - Toxicity / Pathogenicity Testing of  Metarhizium anisopliae
Strain 52 Following Acute Intraperitoneal Challenge in Rats - 
Thirty-nine male and 39 female CD® rats, approximately 51 days old,
were assigned to groups: treatment group (TG), killed treatment group
(KTG), naive control (NC), and shelf control (SC).  The TG group rats
were dosed with approximately 1 ( 107 cfu / mL/animal. Three
rats/sex/group from the TG, KTG, and NC groups were killed and
necropsied on days 0, 3, 7, and 14.  The SC group rats were necropsied
on day 14.  The blood, brain, lungs and associated lymph nodes, spleen,
liver, kidneys, and cecum were removed for microbial enumeration and
weight determination. No adverse signs were observed from any group. No
gross lesions were noted from any rat. Decreased relative spleen, brain,
and liver weights all appeared to be transitory in effect. Based upon
the overall weight gain and lack of pathogenic effects noted upon
necropsy, the differences in organ weights are not considered as
resulting from primary treatment with M. anisopliae F52.  No
statistically significant effects on relative organ weights for other
tissues were observed. Metarhizium anisopliae, Strain F52 does not
appear to be toxic and/or pathogenic in rats, when dosed
intraperitoneally at approximately 1 ( 107 cfu/animal.

Classification: Acceptable.

448447-13 - Primary Eye Irritation Study of Metarhizium anisopliae,
Strain F52, in Rabbits - Three male and three female New Zealand white
rabbits were treated with test material (0.1 g with 6.3 x 108
cfu/eye/animal), applied in the everted lower lid of the right eye, and
the eye held closed for approximately 2 seconds.  The eyes were rinsed
with lukewarm water 24 hours after test material instillation. The
contralateral eye served as control. The eyes were examined and scored
1, 24, 48 and 72 hours and 4, 7, 10, 14, 17, and 21 days after test
material instillation. No deaths were observed in any of the dosed test
animals. Excluding one rabbit because of an unrelated infection, three
rabbits had corneal opacity 24 hours after test material instillation. 
Four rabbits had iritis and five rabbits had positive conjunctival
irritation one hour after test material instillation.  By day 4,  no
irritation was noted on any rabbits with the exception of the male
tagged as No. 497. Since rabbit No. 497 was checked for infection by
swabbing and culture onto TSA and Sabouraud media and no M. anisopliae
was recovered, the infection in this animal is considered to be
unrelated to treatment with the test substance.  Eliminating rabbit No.
497 from the calculations, the highest primary eye irritation score was
23.2 at 24 hours after test material instillation. The lack of test
substance in the rabbit No. 497 indicates the secondary infection
responsible for the substance was not  primarily caused or associated
with treatment with M. anisopliae F52.  Metarhizium anisopliae Strain
F52 at 0.1 g/animal (6.3 x 108 cfu/animal) was moderately irritating
causing corneal opacity, iritis, and conjunctival irritation with
resolution by day 4. 						

Classification: Acceptable.  Toxicity category III for eye irritation.

448447-15 - Dermal Sensitization Study of Metarhizium anisopliae, Strain
F52, in Guinea Pigs Using the Buehler Method - The backs of 40 male
Hartley guinea pigs were clipped one day prior to each treatment.  For
the induction phase, 0.3 g of the test material (2.37 x 109cfu
Metarhizium anisopliae Strain F52) was applied with an occlusive 25 mm
Hilltop Chamber for six hours once each week for three weeks to the
pre-moistened clipped skin of 20 test animals. Two weeks after the third
induction, the animals were challenged with 0.3 g of test material under
occlusion to naive sites for 6 hours. A naive (negative) control group
(10 animals) was treated with 0.3 g of test material at challenge (i.e.,
two weeks after application of the third induction dose) only at the
same site as the treated guinea pigs (i.e., lower left quadrant). The
ten positive control animals were induced with 0.3 mL of
alpha-hexylcinnamaldehyde (HCA) and challenged with 0.3 mL of 50 % w/v
HCA in acetone. All wrapping materials were removed 6 hours after the
challenge dose and the test site rinsed with water and wiped with gauze.
Reactions were scored 24 and 48 hours after the first induction and
challenge. No deaths were observed in any of the guinea pigs. The
animals had normal body weight gains. After three consecutive
inductions, 1/20 treated test animals had a positive reaction 48 hours
after challenge.  No reactions were noted on any naive control animal. 
As for the positive control animals, 6/10 and 7/10 animals had positive
reactions at 24 and 48 hours, respectively, after challenge.  Based upon
the submitted data, Metarhizium anisopliae Strain F52 does not appear to
be a dermal sensitizer when induced and challenged at 2.37 x 109 cfu.

Classification: Acceptable. MaF52 is not considered a dermal sensitizer.

 PAGE  5