IN VIVO MAMMALIAN CYTOGENETI CS –ERYTHROCYTE MICRONUCLEUS ASSAY IN MICE (MRID 47463708)

870.5395 Technical (OMI-88)

In Vivo Mammalian Cytogenetics - Erythrocyte Micronucleus Assay 47463708 Negative. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment

time.

EXECUTIVE SUMMARY : In a bone marrow micronucleus assay (MRID 47463708), 5 or 8 male CD-1 mice/dose were treated twice (24 hrs ap art) via gavage (10 mL/kg) with Tolfenpyrad (99.5% a.i., Lot No. 365-65A) in 0.5% carboxymet hylcellulose at doses of 0, 5, 10, 20, or 50 mg/kg. Due to excessive mortality at 50 mg/kg (6/8 animals died after dosing on Day 1), an additional group of 5 male mice was dosed at 5 mg/kg, and 20 mg/kg became the high-dose.

Bone marrow cells were harvested at 24 hours after the final dosing. Cyclophosphamide (80 mg/kg) served as the positive control. It should be noted that only males were tested with the parent compound because no relevant differences in acute toxicity was seen between the sexes. Accordingly, the requirement for testing in both sexes is waived.

At 20 mg/kg, one mouse was found dead after treatment on Day 1, and one mouse was found dead after treatment on Day 2. No significant decreases in the PCE:NCE ratio were observed in any treatment group compared to controls, indica ting that the test mate rial did not markedly inhibit the growth of bone marrow cells. No trea tment-related increases in the MPCE frequency were observed in any treatment group when compared to controls. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

Although there was no indication of bone marro w growth inhibition, the animals were sufficiently dosed as indicated by the mortality and clinical signs of toxicity observed at 20 mg/kg. Therefore, this study is classified as acceptable/guideline and satisfies the guideline requirement (OPPTS 870.5395; OECD 474) for in vivo cytogenetic mutagenicity data.

COMPLIANCE : Signed and dated Data Confidentiality, GLP Compliance, Flagging, and Quality Assurance statements were provided.

IN VITRO GENE MUTATION ASSAY IN MOUSE LYMPHOMA CELLS (MRID 47463706)

EXECUTIVE SUMMARY : In a mammalian cell gene mutation assay at the TK +/- locus (MRID 47463706), L5178Y mouse ly mphoma cells cultured in vitro were exposed to Tolfenpyrad (99.5% a.i., Lot No. 365-65A) in dimethyl sulfoxide (DMSO) for approximately 4 hours at the following concentr ations: 0, 0.01, 0.1, 1, 5, 10, 25, 50, or 75 μg/mL (Trial 1, -S9); 0,

Page 2 of 30.05, 0.1, 0.5, 1, 10, 25, 75, or 100 μg/mL (Trial 1, +S9); 0, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, or 10 μg/mL (Trial 2, -S9); and 0, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, and 25 μg/mL (Trial 2, +S9). The S9

fraction was derived from the livers of male Sprague-Dawley rats induc ed with Aroclor 1254TM. Methylcholanthrene and methyl methanesulfonate served as positive controls in the presence and absence of S9, respectively.

Tolfenpyrad was tested up to severely cytotoxic (i.e., ≤ 20% relative growth) concentrations ( ≥ 25 μg/mL (+/-S9). No precipitation of the test mate rial was observed at any concentration tested in the presence or absence of S9 in either definitive trial. None of the analyzed concentrations induced mutant frequencies that exceeded the minimum criteria for a positiv e response. In fact, the mutant frequencies generally decreased with increasing concentration both in the presence

and absence of S9-activation in both trials. The positive controls induced the appropriate response both in the presence and absence of S9. There was no evidence of induced mutant colonies over background in the presence or absence of S9-activation. The study is classified as acceptable/guideline and satisfies the guideline requirements (OPPTS 870.5300, OECD 476) for in vitro mutagenicity (mammalian fo rward gene mutation) data.

COMPLIANCE : Signed and dated Data Confidentia lity, GLP Compliance, Flagging, and Quality Assurance statements were provided.

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