Comparative Efficacy of Two Fipronil
`Spot-on Formulations Against Experimen-
`tal Flea Infestations (Ctenocephalidesfelis)
`In Dogs
`
`Stéphane Bonneau, MD Parasitology‘
`
`Josephus J Fourier, MSC2
`
`Carine Rousseau, MD Biostatistics‘
`
`Marie-Christine Cadiergues, DrMedvet, DiplECVD, PhD, MRCVS3
`
`' Virbac SA, BP27, 06511 Carras, France
`
`’C’Iin Vet International (Ply) Ltd,
`PO Bar 11 186, Univezsitas 9321, South Africa
`
`’Groupe de Recherche Animawc de Compagnie,
`Ecole Naiionale Vétérinaire de Ibulouse
`23, chemin des Capelles, 31076 Ibulouse cedex3, France
`
`~
`
` on, flea,
`
`_
`
`T’
`
`ABSTRACT
`
`A new fipronil-based spot-on formulation
`applied once to dogs was evaluated against
`experimental flea infestations in a parallel
`group design, randomized, unicentre and
`blinded controlled study. Eight dogs served
`as negative controls (group 1), eight dogs
`were treated with a 10% W/v fipronil-based
`spot—on solution (Efiipro° Spot—on, Virbac
`SA) at a dosage of 0.67 mL for a dog weigh-
`ing from 2 to 10 kg and 1.34 mLfor a dog
`weighing from 10.1 to 20 kg (group 2) and
`eight dogs served as positive controls (group
`3) receiving the original tipronil spot-on
`(Frontline" Top spot, Merial) at a similar
`dosage. Each dog was infested with 100
`unfed adult CtenocephaIia'esfe1z's on days
`-6, -l. 7. 14. 21. 28, 35, 42. 49, 56. 63, 70,
`77, 84 and 91. At 48 h after treatment or re-
`infestation, each dog was combed to remove
`and count live fleas. Geometric mean flea
`
`2, compared to the negative control group.
`Dogs were protected from re-infestations
`with an efiicacy >95 per cent for 93 days
`in group 2 and for 79 days in group 3. Both
`10% W/v fipronil-based spot-ons, despite
`diflerent vehicles, were equally able to
`eradicate flea infestation, to prevent new
`infestations and were well-tolerated.
`
`INTRODUCHON
`
`Flea infestation remains one ofthe most
`
`frequent ectoparasitic conditions of dogs
`and cats.“ Introduced in 1994, fipronil has
`been a leader on the flea market products for
`dogs and cats ever since. First available as a
`0.25—percent spray? fipronil was then mar-
`keted as a spot-on formulation.‘ and eventu-
`ally was combined with S-methoprene.’ also
`in a spot-on. More recently, pyriprole from
`the same chemical group had been launched
`and is available as a spot-on.” Finally, as
`fipronil’s patent has recently expired in some
`countries. new fipronil-based products are
`now present on the market.
`
`counts obtained were reduced by 99.7 and
`100% in groups 2 and 3. respectively on day
`
`The present study was conducted to
`evaluate the immediate and sustained ef-
`
`] 6
`
`Pb}. 8, No. I, 2010 - Intern JAppl Re: Vet Med.
`
`Exhibit 1014A
`
`Merial v. Virbac
`
`IPR2014-01279
`
`

`
`ficacy and the tolerance of a new spot-on
`formulation (Effiprog Spot-on. Virbac S.A.)
`with the same qualitative and quantitative
`composition in terms of active ingredient
`(fipronil) as the original product (Fmut-
`line" Top spot. Merial), but with different
`vehicles. The eflicacy was evaluated against
`experimental infestations with Ctenoce-
`phalidesfelis in dogs. Apositive reference
`control group included dogs treated with the
`original product.
`MATERIALS AND METHODS
`
`Twenty four mongrel dogs (17 female _and
`7 male) over 4 months of age and weighing
`from 7.2 to 17.9 kg were initially included
`in the study. Three dogs (one male and two
`female) belonging to the negative (untreat-
`ed) control group were replaced on Day 48
`with three female dogs. Dogs were allocated
`randomly into three groups of eight and
`each dog was individually housed in a 1.9 x
`2.7-meter pen. They were fed a commercial
`dog diet and water was available ad Iibimm.
`They were acclimatized for 7 days prior to
`treatment.
`
`A laboratory bred strain of Ctenocephal-
`idesfelis (C1inVet-2004). routinely fed on
`cats was used for all infestations. For each
`
`experimental infestation, approximately 100
`unfed. young adult fleas of mixed sex were
`placed on each dog’s dorsum. The dogs were
`infested six and one day prior to the treat-
`ments and 7. 14, 21. 28. 35. 42. 49, 56. 63.
`70, 77. 84 and 91 days after the treatments.
`
`The study was a parallel—ann, ran-
`domized block design. unicentre, blinded
`controlled study. The animals were not
`treated by an individual involved in the post-
`treatment assessments and observations.
`
`Study groups were coded to blind the staff
`performing post-treatment assessments and
`observations. The dogs were ranked within
`gender in descending order of individual
`body weight. Within each gender. animals
`were then allocated to blocks of three dogs
`each. Vfithin each block, dogs were random-
`ly allocated to groups 1 to 3.
`The dogs in group 1 were not treated.
`The dogs in group 2 were treated with a 10%
`
`w/v fipronil-based spot-on solution (Effipro°
`Spot-on. Virbac SA) at a dosage of 0.67 n1L
`for a dog weighing from 2 to 10 kg and 1.34
`mL for a dog weighing from 10.1 to 20 kg.
`The dogs in group 3 were treated with the
`original fipronil spot—on (Frontline‘’ Top
`spot. Merial) at a similar dosage. The solu-
`tion used in group 2 had the same qualitative
`and quantitative composition in terms of
`active ingredient (fipronil) as Frontline” Top
`spot but some vehicles were different. Both
`products were applied topically as a single
`spot on the skin between the shoulders. Care
`was taken to avoid wetting the hair or ap-
`plying the dose to an area where the animal
`could lick it off. Dogs were restrained for
`one minute afler dosing. Concurrent treat-
`ments unlikely to interfere with the study
`were acceptable (antimicrobials. vitamins
`and mineral supplements and sedatives) and
`the treatment details were recorded. Sub-
`
`stances that may have had an insecticidal or
`acaricidal activity (e. g. medicated sham-
`poos) were not allowed.
`Each animal was submitted to a full
`
`clinical examination on days -7. 27 and 63.
`Additionally on day 48. a full clinical ex-
`amination was conducted on the three dogs
`replacing the three dogs withdrawn from
`the study in the negative control group. Any
`adverse event was recorded.
`
`Forty-eight hours afier the treatment and
`48 hours after each challenge, the popula-
`tion of remaining fleas was assessed for each
`animal. Three operators were involved in the
`assessment of a specific animal. One person
`handled and restrained the dog, a second
`was responsible for combing the dog and a
`third person was responsible for quantify-
`ing the fleas recovered from each comb
`and recorded the data. During combing. a
`fine-toothed flea comb was used to recover
`
`'
`
`fleas present in the dogs fur. The method
`of combing included several strokes of the
`comb in each area of the animal. each time
`
`moving in the same direction. following the
`pattern of the hair coat. Movement from
`one part of the dog‘s fur to the next was Via
`strokes overlapping each other. so that no
`
`Intern JAppl Res I/tat Med - Vol. 8, No. I, 2010.
`
`17
`
`

`
`area of fur was missed. After completion of
`the combing procedure for all body areas.
`the whole procedure was repeated so that all
`areas were combed twice. All fleas collected
`
`were cormted and were not replaced on the
`animals.
`
`Analyses were performed with the com-
`mercial software SAS V9.1. For all analyses,
`the significance threshold was set to (1 =
`0.05. The three groups were described and
`compared before treatment (baseline) on the
`following criteria: weight. sex, average hair
`length and flea count. Qualitative parameters
`were analyzed using a Fisher‘s exact test
`and quantitative parameters were analyzed
`using a Kruskal-Wallis test. Arithmetic and
`geometric means were calculated for each
`of the three groups at each time point. For
`groups 2 and 3. efiicacy was calculated at
`each time point using the mean according
`to Abbott‘s formula: Eflicacy (%) = 100 X
`(mean mm —rnean mm)! mean mm. Flea
`count was analyzed by a one-way analysis
`of variance on ranked data.. The p-value of
`the comparison between groups 2 and 3 was
`given and compared to 0.017 (Bonferroni
`correction for multiple comparisons).
`
`RESULTS
`
`The three groups were homogenous at base
`line on the following criteria: weight. sex.
`average hair length and flea count (Table 1).
`Three dogs from the control group devel-
`oped severe signs consistent with flea allergy
`derrnatitis (FAD), so they were excluded
`and replaced with three new dogs on day 48.
`Two other dogs. one from group 1 and one
`from group 3 developed similar signs and
`were excluded on day 79 and not replaced.
`No adverse events that could be related to
`
`the administration of either product were
`recorded in any of the treatment groups.
`The arithmetic mean numbers of fleas
`
`that were present in the hair coat of the un-
`treated control dogs and on treated animals
`48 hours after each infestation are graphical-
`ly illustrated in Figure 1. The efficacy. based
`on geometric means. of both formulations of
`fipronil is summarized in Table 2.
`The results (Figrne 1 and Table 2) Show
`that the experimental infestations with C.
`jélis were successful, with a mean percent-
`age recovery of C. felis on the control
`dogs 48 hours after each infestation rang-
`ing between 51 (day 9) and 88.4 (day 51)
`
`Figure 1. Aritlnnetic mean (sci) Ctenoceplmlidesfelis counts 48 hours afier treatment‘ with
`mo 10% w/\*fipronil—based spot-on solutions on day 0 and 48 hours afler weekly re—infesr(r—
`fion with I 00 mgfed adultfleas over 13 weeks.
`
`I20
`
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`llunmrdterrof §
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`4”
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`—-—-Control ---Effipm® —fi—frontline®
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`29 06
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`23
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`30
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`37
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`4‘!
`
`SI
`
`58
`
`65
`
`72
`
`19
`
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`
`93
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`Buy: «fur trutmmt
`
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`ta]. 3, No. 1, 2010 - Inrem JAppI Res’ Vet Med. '
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`

`
`Table 1. Means (standard deviation; n=8), median, minimal and maximal values and P-values
`(using a Kruskal— Wallis test) ofweight, average hair length andpre-treatmentflea countfimn
`(logs treated with one ofthe two 10% 1t’/\‘fipI‘0l1fl-b(YS€(l spot-on solutions or left untreated.
`3
`edi
`73E”
`Mean (st
`0.8331
`9.30
`10.20 (2.39)
`10.45
`10.48 (2.21)
`10.30
`21.0
`16.3
`
`7.80-14.90
`
`7.SO—14.6O
`720-1790
`17.5-28.8
`
`11.8-44.5
`95-27.5
`
`Illlllllli
`
`72.5
`78.5
`73.0
`
`52.0~86.0
`58.0-92.0
`27.0-94.0
`
`
`
`
`
`
`03326
`
`0.3677
`
`Pre—treat:me'nt
`
`Negative control
`
`flea °°"”‘
`
`Effipro‘
`Etline‘
`
`69.9 (11.9)
`
`73.0 (9.3)
`69.8 (22.3)
`
`Weight (kg)
`
`
`
`Treatment emu»
`Negative contml
`
`
`
`Eflipxo‘
`I-'ronfline'
`— u 24 (3.36)
`21.3 (3.4)
`
`Average hair
`1°"3"’ (mm)
`
`
`
`21.2 (11.8)- 17~o<s-9>
`
`Table 2. Mean geometric efiicacy (96) of two 10% w/vfipronil-based spot-on solutions applied
`to dogs experimentally infizsted with Ctenocephalides felis, calculated 48 hours after the treat-
`ment and 48 hours after each weekly re-infestation over 13 weeks.
`
`
`
`ZINE!
`IEEIEIME 992
`97-1
`I'.EEII1‘§IIEIII§IE§I%E 75-4
`
`Days after treatment
`
`percent. The application of the two formula-
`tions of fipronil led to the complete eradica-
`tion of all fleas but two (collected from one
`dog in group 2) when dogs were examined
`on day 2. The mean therapeutic efficacy was
`99.7 percent in group 2 and 100 percent in
`group 3. The difference between the two
`groups was not statistically significant (p =
`0.35). Dogs in group 2 were protected from
`re-infestations for 93 days with an efficacy
`of at least 95 percent. Dogs in group 3
`were protected from re—infestations for 79
`days with an efficacy of at least 95 percent.
`Efficacy was further reduced to 88.5 per
`cent on day 86 and 75.4 percent on day 93.
`Differences between the two groups were
`significant on days 86 and 93 (p = 0.0001
`and 0.0023, respectively).
`DISCUSSION
`
`Both products were well tolerated by all
`the animals that received them. A total of
`
`five dogs were excluded from the study as
`they developed severe clinical signs con-
`sistent with FAD. Four were untreated and
`
`one had received Front1ine‘° Top spot. It is
`understandable that an individual. possibly
`hypersensitive to fleas, that was treated
`almost three months before. might develop
`severe dennatologic signs following a mas-
`sive infestation (IOO unfed fleas). Moreover,
`this enhances the necessity for dogs with
`FAD to be treated drastically. either apply-
`ing a product more frequently than what
`the manufacturer recommends (every 2-3
`weeks). or better combining two difierent
`molecules with a dilferent mode of action.“
`
`Efi’tpro° Spot-on and Frontline° Top spot
`were both very eifective treatinents for flea
`infestation in dogs (efiicacy of 99.7 and 100
`percent respectively on day 2). This level
`of control is comparable to the efiicacy of
`Frontline” Spot—on reported in various simi-
`lar studies against experimental flea infesta-
`tions in dogs” or in semi-field studies."
`Under the conditions of this study. both
`treatments provided long-lasting residual
`protection against flea infestations (93 and
`79 days for Efiipro° Spot-on and Front-
`line° Top spot. respectively). However. the
`
`Intern JAppl Res Vet Med - Vol. 8, No. 1, 2010.
`
`19
`
`

`
`standardized procedures. the absence of
`re-infestations from the environment or from
`
`other animals, the climatic stability, and the
`absence of bathing/swimming or other skin
`interventions. which could impair the diffu-
`sion of the product andfor its persistence on
`the skin. make the conditions of the present
`trial ideal. Therefore, under field conditions.
`such a long-lasting residual protection is
`unlikely. Hence the information on both
`products’ data sheets which indicate that the
`insecticidal efficacy against new infesta-
`tions with adult fleas persists for up to eight
`weeks. Fuithenriore. recommendations of
`
`monthly applications are comnionly made,
`based on previous field studies."~“‘-*5
`
`Both Etfipro° Spot-on and Frontline‘
`Top spot are 10% w/v fipronil—based spot-on
`solutions but some of their vehicles are dif-
`
`ferent. The present study shows that despite
`different vehicles, the two formulations were
`equally able to eradicate flea infestation, and
`prevent new infestations. They were also
`equally well-tolerated.
`R EF E R EN C ES
`1.Faxkas R, Gyurkovszky M, Solymosi N, Beugnet 1-‘:
`Prevalence offlea infestation in dogs and cats in
`Hungary combined with a survey of owner aware-
`ness. Med Vet Entomol 2009;23:187-194.
`2.Rinaldi L, Spent G. Musella V, Carbone S, Veneziano
`V, Iori A, Cringoli G:A survey offims on dogs in
`southern Italy. Vet Parasite} 2007;148:375—378_
`3.Bond R, Riddle A, Mottram L, Belignet F, Steven-
`son R; Survey of flea infestation in dogs and cats
`in the United Kingdom during 2005. Vet Rec
`2007;l60:503—506.
`4.D\mden LA, Judy TN, Martin IE, Spedding LS: Fleas
`parasitizing domstic dogs in Georgia, USA:
`Species composition and seasonal abundance. Vet
`Parasitol 2005;80:157-162.
`
`5.Rust MK: Advances in the control of Ctenocephalidm
`felt’: (cat flea) on cats and dogs. Trends Pamritol
`2005;21:232—236.
`61-'ranc M, Choquart P, Cadiergues MC: Species
`offleas found on dogs in France. RevMed I/kt
`1998;149:135-140.
`7.Postal JMR_ Jezmnin PC, Consalvi PI: Field ef-
`ficacy of a mechanical pump spray fommlation
`containing O_25—percent fipronil in the treatment
`and control offlea infestation and associated
`dermatological signs in dogs and cats Vet Dermatol
`l995;6:l53-158.
`8.A.twe_ll R, Fitzgerald M, Howlett B, Jensen C, John-
`stone 1, Page A, Robatto 1, Postal JM: The use of
`topical fipronil in field studies for flea control in
`domestic dogs. Aux! PEI Pract 1997;27:184-134.
`9.You:ng DR, Ieannin PC, Boeckh A: Eficacy of fipro—
`njl/(S)—methoprene combination q>ot—on for dogs
`against shed eggs, emerging and existing adult can
`fleas (Ctenocephalides felis, Bouche). Vet Pararitol
`2004; 125397-407.
`10.Ba:nett S, Luempert L, Schuele G, Quezada A, Stre-
`hlau G, Doherty P: Eficacy ofpyriprole topical
`solution against the cat flea, Ctenocephalidwfelis,
`on dogs. Vel Ther 2008;9:4-14.
`1l.Cadiergues MC: Flea control in flea allagic dogs
`and cats. EICAP 2010; in press.
`l2_Cadiergues MC, Caubet C, Franc M: Comparison of
`the activity of selamectin, imidacloprid and fipronil
`for the treatment of dogs infested experimentally
`with Ctenocephalides Curtis and Ctenoczphalides
`felisfelir. VatRec 200l;l49:704—706.
`13.Ritzhaupt LK, Rowan TG, Jones RL: Evaluation of
`efficacy of selamectin, fipronil, and imidacloprid
`against Ctenocqahaliderfélis in dogs. JAm Vet
`Medxlssoc 2000;217:1669-1671.
`14.Dryden MW, Denenberg FM, Bunch S: Control of
`fleas on naturally infested dogs and cats and in
`private residences with topical spot applications of
`fiprouil or imidacloprid. Vet Parasitol 2000;93:69-
`75.
`l5.Medleau L, Clekis T, McA.rthur TIL Alva R, Barrick
`RA Jeimnin P, Irwin 1: Evaluation offipronil spot-
`on in the treatment of flea allergic dermatitis in
`dogs. J SmaIlAn1'm Pract 2003;44:71—75.
`
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