## 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 Microbia l 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 L eader Biopesticide s 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 gre enhouse 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 scien tists. 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 worldw ide from insects and non
- insect sites, such as soil, river sediments, associated with nematodes, and as a saprophy te on organic detritus.2

DATA REVIEW RECORD Active Ingredients: Metarhizium anisopliae strain F52 Product Name: TAE
- 001 Technical Bioins ecticide 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 MRI D/Study

Titles: 4 48447
- 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 Followin g Acute Intratracheal Challenge in Rats 448447
- 12
- Toxicity

/ Pathogenicity

Testing of Metarhizium anisopliae Strain 52 Following Acute Intraperitoneal Challenge in Rats 448447
- 13
- Primary

Ey e Irritation Study

of Metarhizium anisopliae, Strain F52, in R abbits 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 day s 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 approxi mately

1.04 x 10 8 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 day s 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 day s 0 , 3, 7, 14, and 21. Due to the clearance of the test material by day 3, the animals scheduled to be sacrificed on day s 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, kidney s, mesenteric ly

mph 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 w as below the detection limit. Metarhizium anisopliae Strain F52 does not appear to be toxic and/or pathogenic in rats,3

when dosed at 1.04 x 10 8 cfu/animal. Classification: Acceptable. 448447 - 10 - Acute Dermal Toxicity / Pathology Study of Metarhizium a nisopliae Strain 52 in Rabbits - Five male and five female New Zealand white rabbits were received from Kuiper Rabbit Ranch, Gary , I

N. 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 10 10 cfu/animal) was applied evenly to the clipped dorsal trunk in an area of approximately 10% (about 240 cm 2 ). The application site was covered with a surgical dressing and a plastic film, then secured with a lint
- f

ree cloth and an elastic adhesive bandage. The skin was checked for irritation within one hour after patch removal and daily thereafter for 14 day s. The rabbits were weighed prior to treatment and on day s 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 ery

thema 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 femal es 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 sy mptoms before the end of the study
. No deaths and no clinical toxicity were observed. The acute lethal dose (LD 50 ) is greater than 2000 mg/kg. Classification: Acceptable - Toxicity category III.
448447 - 11 - Toxicity / Pathogenicity Testing of Metarhizium anisopliae Strain 52 Followi ng Acute Intratracheal Challenge in Rats - Eighty male and 80 female CD ® rats, approximately 70 day s 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  10 8 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 day s 0, 7, 21, and 35. The SC group rats were necropsied on day

35. The blood, brain, lungs and associated ly mph nodes, spleen, liver, kidney s, and cecum were removed for microbial enumeration and weight determination. Mean body weight of males and fema les 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 day s 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 rel ative 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 considere d as not biologically

meaningful and likely

reflects the normal variation present in organ weights of4

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 ly mph nodes of males on day s 0 and 7 and were below the detection limit by day 21. The test organism was detected in the lungs and associated ly

mph nodes of females on day s 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  10 8 cfu/animal. Classification: Acceptable. 448447 - 12 - Toxicity / Pathogenicity Testing of Metarhizium anisopliae Strain 52 Following Acute Intraperitoneal Challenge in Rats - Th irty
- nine male and 39 female CD ® rats, approximately

51 day s 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 approxi
mately 1  10 7 cfu / mL/an imal. Three rats/sex/group from the TG, KTG, and NC groups were killed and necropsied on day s 0, 3, 7, and 14. The SC group rats were necropsied on day 14. The blood, brain, lungs and associated ly mph nodes, spleen, liver, kidney s, 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 overa ll 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 tis sues were observed. Metarhizium anisopliae , Strain F52 does not appear to be toxic and/or pathogenic in rats, when dosed intraperitoneally at approximately

1  10 7 cfu/animal. Classification: Acceptable. 448447 - 13 - Primary Eye Irritation Study of Metarh izium 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 10 8 cfu/ey e/animal), applied in the everted lower lid of the right eye, and the ey e held closed for approximately

2 seconds. The ey es 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 day s after test material ins tillation. 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 Sab ouraud 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 ey e 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 Str ain F52 at 0.1 g/animal (6.3 x 10 8 cfu/animal) was5

moderately

irritating causing corneal opacity , iritis, and conjunctival irritation with resolution by day 4. Classification: Acceptable. Toxicity category III for eye irritation. 448447 - 15 - Derma l 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 10 9 cf u 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 mLof 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 10 9 cfu. Classification: Acceptable. MaF52 is not considered a dermal sensiti zer.