IN VIVO MAMMALIAN CYTOGENETICS –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 apart) via gavage (10 mL/kg) with Tolfenpyrad 

(99.5% a.i., Lot No. 365-65A) in 0.5% carboxymethylcellulose 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, indicating that the test material did not markedly 

inhibit the growth of bone marrow cells. No treatment-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 marrow 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 lymphoma 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 concentrations: 0, 0.01, 0.1, 1, 5, 10, 25, 50, or 75 μg/mL (Trial 1, -S9); 0, 

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0.05, 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 induced 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 material 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 positive 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 forward gene mutation) data. 

COMPLIANCE: Signed and dated Data Confidentiality, GLP Compliance, Flagging, and 

Quality Assurance statements were provided. 

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