Evaluation of the acaricidal activity of thymol ... - Springer Link

Parasitol Res DOI 10.1007/s00436-017-5604-x

ORIGINAL PAPER

Evaluation of the acaricidal activity of thymol incorporated in two formulations for topical use against immature stages of Rhipicephalus sanguineus sensu lato (Latreille, 1806) (Acari: Ixodidae) Camila Delmonte 1 & Paula Barroso Cruz 2 & Viviane Zeringóta 2 & Valéria de Mello 3 & Felipe Ferreira 3 & Maria da Penha Henriques Amaral 3 & Erik Daemon 1

Received: 10 May 2017 / Accepted: 27 August 2017 # Springer-Verlag GmbH Germany 2017

Abstract The objective of this study was to assess, for the first time, the in vitro acaricidal activity of two topical formulations containing thymol, on immature stages of Rhipicephalus sanguineus sensu lato. For this purpose, two base formulations were prepared: an oil-in-water (O/W) emulsion and a hydroalcoholic solution, containing different thymol concentrations (0.5 to 20 mg/mL). We used the larval packet test for non-engorged larvae and nymphs, and the immersion test for engorged larvae and nymphs. For emulsion, a mortality rate of 94.2% was achieved at 0.75 mg/mL in nonengorged larvae. For engorged larvae, there was 95.0% mortality at 5.0 mg/mL. Non-engorged nymphs showed 83.3% mortality at 2.5 mg/mL, and for engorged nymphs, 86.0% mortality was verified at 5.0 mg/mL. For the hydroalcoholic solution, the mortality found for non-engorged larvae was 88.1% at 2.5 mg/mL. For engorged larvae, the highest mortality was 25.0% at 20 mg/mL; non-engorged nymphs had 91.0% mortality at 1.0 mg/mL and for engorged nymphs; the maximum value verified was 18.3% mortality at 20 mg/ mL. Preliminary stability tests were carried, and the hydroalcoholic solution remained stable under all the * Camila Delmonte [email protected]

1

Programa de Pós Graduação em Ciências Biológicas Comportamento e Biologia Animal, Universidade Federal de Juiz de Fora UFJF, Juiz de Fora, Minas Gerais, Brazil

2

Programa de Pós Graduação em Ciências Veterinárias, Departamento de Parasitologia Animal, Universidade Federal Rural do Rio de Janeiro, UFRRJ, Seropédica,, Rio de Janeiro, Brazil

3

Programa de Pós Graduação em Ciências Farmacêuticas, Universidade Federal de Juiz de Fora, UFJF, Juiz de Fora, Minas Gerais, Brazil

conditions analyzed. The O/W emulsion showed signs of early instability at the concentration of 5.0 mg/mL. The results obtained indicate that the acaricidal activity of thymol, when included in the proposed formulations, was enhanced against non-engorged larvae with topical treatment in comparison with data in the literature. Although there were variations in toxicity between the different stages, these formulations are promising for future therapeutic use. Keywords Brown dog tick . Monoterpene . Larvae . Nymphs . Emulsion . Hydroalcoholic solution

Introduction Rhipicephalus sanguineus sensu lato (Latreille, 1806), the brown dog tick, is widely distributed in the world (Labruna 2004; Guglielmone et al. 2006). The control of this ectoparasite should take into account that it is a three-host tick, which means that each stage of its life cycle must feed on a host, and that oviposition and molts are made in the environment. Besides the direct damage caused, such as blood spoliation, discomfort, and allergic reactions, it can also act as a vector for dissemination of bacteria and protozoa to dogs and humans (Dantas-Torres 2008). In Brazil, it is considered a potential vector of human infection by the bacterium Rickettsia rickettsii (Serra-Freire et al. 2011). The taxonomic status of this species still lacks consensus. In recent years, evidence of genetic, morphological, and epidemiological differences of individuals from different regions of the world has been reported, and based on the difficulty of determining the neotype R. sanguineus sensu stricto, it has been established that the most suitable denomination for

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individuals of this complex is Rhipicephalus sanguineus sensu lato (s.l.) (Nava et al. 2015). Control of these parasites is usually by applying synthetic chemical substances. However, the indiscriminate use of these substances causes selection of resistant tick populations, intoxication of animals and their handlers, and possible contamination of the environment with chemical wastes (Chagas 2004; Coles and Dryden 2014). Since the relationship between humans and domestic animals has been getting increasingly close, with dogs often considered as members of the family, the importance of parasite control and the use of safe chemicals is emphasized, in order not to compromise the health of pets and their guardians. These drawbacks have resulted in growing demand for safer acaricides, in turn, prompting research into substances derived from plants (Chagas, 2004; Ellse and Wall 2014; Regnault-Roger and Philogène, 2008; Borges et al. 2011). Among the substances of plant origin already tested for this purpose is thymol, an aromatic monoterpene initially isolated from plants of the family Lamiaceae. This molecule has shown promising results regarding its potential against various species of ticks (Monteiro et al. 2010; Mendes et al. 2011; Daemon et al. 2012; Matos et al. 2014; Araújo et al. 2015; Novato et al. 2015). For a chemical substance, whether natural or artificial, to be used for therapeutic purposes, it is often necessary to include it in a formulation that makes its application feasible and does not impair its efficacy (York 2005). Therefore, the aim of this study was to assess the in vitro acaricidal activity of thymol, incorporated in two formulations for topical use, against immature stages of R. sanguineus s.l., along with the formulations’ short-term stability.

placed in plastic syringes with the distal end cutoff, sealed with hydrophilic cotton, and labeled for identification. The syringes were kept under the same temperature and humidity conditions, and the larvae were used in the tests 15–25 days after hatching. The unengorged nymphs, derived from engorged larvae, were tested 15 days after ecdysis and the engorged larvae and nymphs, obtained by artificial infestation on rabbits, were tested on the day they dropped off the host. Formulation development Two base formulations were prepared, one an oil-in-water (O/W) emulsion and the other a hydroalcoholic solution. The thymol utilized in the formulations was obtained from Sigma-Aldrich, with purity ≥ 99%. The O/W emulsion was formulated with the following raw materials, identified according to the International Nomenclature of Cosmetic Ingredients (INCI): cetearyl alcohol, sodium laureth sulfate, metylparaben, propylparaben, glycerin, grape seed oil, and water. For preparation, each constituent was separated according to its solubility in water or oil. The aqueous phase was heated to 80 °C and the oil phase to 75 °C, and then the aqueous phase was added in the oil phase and emulsified with a mortar and pestle until completely homogenized. The hydroalcoholic solution contained glycerin, methylparaben, and ethanol and was prepared by mixing the components at room temperature in a graduated glass beaker. Twenty-four hours after preparation, the samples were evaluated regarding homogeneity and organoleptic characteristics, to identify any instability. In both formulations, the thymol, dissolved in absolute ethanol q.s. (sufficient quantity), was added after preparation of the base formula. Bioassays

Material and methods Study location The formulations were prepared, and their stability was analyzed in the Laboratório de Farmacotécnica da Universidade Federal de Juiz de Fora, Minas Gerais, Brazil. The in vitro tests on ticks were performed in the Laboratório de Artrópodes Parasitos of the same University. Origin of the ticks The R. sanguineus s.l. larvae used came from a colony maintained by an artificial infestation of rabbits (Oryctolagus cuniculus Linnaeus, 1758—New Zealand x California cross), according to the method proposed by Neitz et al. (1971). The engorged female ticks were kept in a climate-controlled chamber (27 ± 1 °C and RH 80 ± 10%) for oviposition. After 15 days, the eggs were weighed into aliquots of 200 mg,

For the in vitro tests, 18 groups were formed, nine for the emulsion and nine for the hydroalcoholic solution: emulsion (or solution) control, and emulsion (or solution) with thymol at 0.5, 0.75, 1.0, 1.25, 2.5, 5.0, 10.0, and 20.0 mg/mL. These concentrations were chosen based on tests previously carried out with thymol on R. sanguineus s.l. at concentrations ranging from 2.5 to 20 mg/ mL (Daemon et al. 2009; Monteiro et al. 2009; Daemon et al. 2012; Senra et al. 2013), and lower thymol concentrations were also tested to assess possible enhancement of the acaricidal effect caused by the interaction of thymol with the adjuvants in the formulations. The larval packet test was used for the unengorged phases, as proposed by Stone and Haydock (1962) and adapted by Monteiro et al. (2012). Approximately 100 larvae were placed on sheets of filter paper (6 × 6 cm), which were then folded and sealed with three binder clips. Then each side of the filter paper envelope was moistened with 90 μL of the formulation to be tested, and the envelopes containing the ticks were placed in a climate-controlled chamber (27 ± 1 °C and RH 80 ± 10%). Each

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envelope corresponded to a sample unit, and each treatment was repeated ten times. The envelopes were opened after 24 h to count the number of live and dead larvae, using a vacuum pump attached to a hose to collect the live ones. The average mortality was expressed as a percentage, according to the following formula: mortality (%) = (total dead larvae/total of larvae) × 100. The same method was used for the unengorged nymphs, except only five nymphs were placed in each packet. The engorged larvae were submitted to the immersion test, as proposed by Drummond et al. (1973). Larvae recovered from the artificial infestation of rabbits were separated into groups of 100 individuals, placed in 10-ml beaker and immersed in 5 ml of the formulations for 5 min, with each group corresponding to a treatment. After immersion, the larvae were dried on paper towels and divided into subgroups of ten each, which were placed in labeled test tubes and sealed with hydrophilic cotton, so that each tube corresponded to a repetition. Therefore, ten repetitions were performed for each treatment, with each repetition containing ten engorged larvae. The mortality rates were assessed after 15 days, during which period the tubes were kept in a climate-controlled chamber at 27 ± 1 °C and RH 80 ± 10%. The average mortality percentages were calculated from the same equation used for the unengorged larvae. The same technique was used for the tests with engorged nymphs, except each experimental unit contained five nymphs, and each treatment was repeated ten times. Data analysis The Bioestat version 5.0 software was used for statistical analysis. The percent data of mortality were transformed (√arcsin x) and analyzed by the Kruskal-Wallis and Student-NewmanKeuls tests (p < 0.05). For the evaluation of pH averages, Anova and Tukey’s tests were adopted. Preliminary stability tests The preliminary stability of the formulations was evaluated by accelerated or short-term stability testing (Brasil 2004). In this test routine, the formulations were stored for 15 days under different stress conditions, to accelerate possible instability. The samples were initially evaluated 24 h after preparation, by sensory assessment, centrifugation and pH measurement, and again after 15 days of stress. The storage conditions were (1) high temperature (in an oven at 37 ± 2 °C), (2) low temperature (in a refrigerator at 5 ± 2 °C), (3) room temperature (25 ± 2 °C), with and without light exposure, and (4) freeze-thaw cycles (24 h at 40 ± 2 °C and 24 h at 4 ± 2 °C, alternately). These tests were only performed on the emulsion and hydroalcoholic solution at concentrations of 2.5 and 5.0 mg/ mL, because these were the smallest concentrations that presented satisfactory acaricidal activity on more than one stage

of ticks tested. All the tests were performed in clear graduated polypropylene tubes, properly sealed. The sensory evaluation involved judging changes in color, odor, and appearance, after subjecting the samples to stress. The centrifugation was conducted on 10 mL of each sample, at 1509 g for 30 min at room temperature, followed by observation of the presence or absence of macroscopic signs of instability, such as creaming, coalescence, and flocculation. The pH was measured in with a potentiometer after calibration with buffer solutions at pH 4 and pH 7, on samples diluted 1:10 in recently distilled water, at room temperature (25 ± 2 °C). Each sample was measured three times, and the results presented correspond to the mean ± standard deviation of the three readings. The sensory characteristics were ranked in three categories: normal, without alteration (N); slightly modified (SM); and intensely modified (IM) (Brasil 2004).

Results In the tests with the emulsion formulations on unengorged larvae, all the groups showed mortality near 100% starting at the concentration of 0.75 mg/mL, in all cases, significantly different than the control group. For engorged larvae, higher thymol concentrations were necessary to reach relevant mortality rates, which peaked at 95% for the concentration of 5.0 mg/mL. In turn, in the test with unengorged nymphs, rising mortality rates were observed as the thymol concentration increased, with rates of 83.3% for the concentration of 2.5 mg/mL, rising to 100% with concentration of 20 mg/mL. In the test with engorged nymphs, the average mortality was 86% at the concentration of 5.0 mg/mL (Table 1). In the hydroalcoholic solution tests, the concentration of 2.5 mg/mL caused 88.1% mortality on unengorged larvae and reached 98.1% at 5.0 mg/mL, and 100% at 10.0 and 20.0 mg/mL. The activity against engorged larvae was partial, since the highest concentration tested (20 mg/mL) only caused 25.0% mortality, a value considered too low for effective tick control (Brasil 1997). In the test with unengorged nymphs, mortality of 91% was achieved at the concentration of 1.0 mg/mL, reaching 100% from 5.0 mg/mL onward, while in the case of the engorged nymphs, the hydroalcoholic solution did not present relevant acaricidal activity, even at the highest concentrations, attaining maximum mortality of only 18.3% with the highest concentration (20 mg/mL) (Table 2). When submitting the O/W emulsion and hydroalcoholic solution at the concentrations of 2.5 and 5.0 mg/mL to centrifugation in the initial stability triage, we noted that the emulsion at 5 mg/mL was intensely modified, with signs of coalescence. This sample was, thus, excluded from the subsequent stability studies. The O/W emulsion at 2.5 mg/mL, and the hydroalcoholic solution at both concentrations tested

Parasitol Res Table 1 Mortality rates (mean ± SD) of immature stages of Rhipicephalus sanguineus sensu lato treated with different concentrations of thymol incorporated in a O/W emulsion under laboratory conditions (27 ± 1 °C and RH 80 ± 10%)

Treatments

Unengorged larvae

Engorged larvae

Unengorged nymphs

Engorged nymphs

Control

0.8 ± 1.4a

4.0 ± 8.4ª 12.5 ± 16.3ab 41.7 ± 17.5b 41.7 ± 15.7b 40.2 ± 25.2b

4.0 ± 8.4a 3.3 ± 10.5a 2.0 ± 6.3a 2.0 ± 6.3a 0.0 ± 0.0a

83.3 ± 22.1c 86.7 ± 12.4c 97.2 ± 8.3c 100.0 ± 0.0c

32.0 ± 23.5b 86.0 ± 21.2bc 95.0 ± 9.3bc 100.0 ± 0.0c

0.5 mg/mL 0.75 mg/mL 1.0 mg/mL 1.25 mg/mL

73.2 ± 11.6 94.2 ± 9.8bc 100.0 ± 0.0c 99.5 ± 1.3c

3.0 ± 4.8ab 0.0 ± 0.0a 5.0 ± 7.1ab 16.0 ± 7.0bc 29.0 ± 14.5c


100.0 ± 0.0c 100.0 ± 0.0c 100.0 ± 0.0c 100.0 ± 0.0c

40.0 ± 15.6cd 95.0 ± 5.3de 100.0 ± 0.0e 100.0 ± 0.0e

ab

Averages followed by equal letters in the same column do not differ statistically at a significance level of 5%. Control = emulsion without thymol

did not show alterations after initial centrifugation, so they were submitted to the storage challenge tests. After exposure for 15 days to the different temperature and lighting regimes, all the samples continued to have stable color and characteristic thymol odor. The hydroalcoholic solution was most stable in all the situations and at both concentrations. The O/W emulsion at 2.5 mg/mL remained stable at room temperature, with and without exposure to light, and when kept under continuous refrigeration; but it showed signs of instability (creaming) after the final centrifugation in the samples maintained in the oven and subjected to the freeze-thaw cycle. The measurement of pH demonstrated a non-significant variation (p < 0.05) between the initial values (1st day) and final ones (15th day) for all the formulations tested, according to the ANOVA and Tukey test. The results of the sensory analysis, centrifugation, and pH measurement are shown in Table 3.

Discussion For many years synthetic substances were used with good results to control ticks. However, due to their frequent Table 2 Mortality rates (mean ± SD) of immature stages of Rhipicephalus sanguineus sensu lato treated with different concentrations of thymol incorporated in a hydroalcoholic solution under laboratory conditions (27 ± 1 °C and RH 80 ± 10%)

indiscriminate use and physicochemical characteristics, today the occurrence of selection of resistant populations is becoming more common. Additionally, these substances cause environmental contamination and intoxication of animals and their owners. Thus, it is necessary to find substances that are effective but have less impact on human and animal health and the environment, besides imposing less selective pressure for the resistance of ticks. Thymol has been studied for several years as a bactericide, fungicide, acaricide, and insecticide (Carvalho et al. 2003; Floris et al. 2004; Botelho et al. 2007; Vasconcelos et al. 2014), and has been classified as a safe substance by the US Environmental Protection Agency (USEPA 1993). One of the reasons for this safety is that it quickly dissipates in the environment, leaving low residue levels (Hu and Coats 2008). The results obtained in this study showed a stronger acaricidal effect of thymol on unengorged larvae in the tests with the emulsion and hydroalcoholic solution compared to the results reported by Daemon et al. (2009) and Daemon et al. (2012). In the case of the engorged larvae, the emulsion was more toxic. Indeed, the hydroalcoholic solution did not cause relevant mortality rates, reaching levels lower than those obtained when thymol was tested diluted in water + DMSO at

Treatments

Unengorged larvae

Engorged larvae

Unengorged nymphs

Engorged nymphs

Control

0.1 ± 0.3a

0.0 ± 0.0ª 4.5 ± 9.6ª 5.0 ± 10.0ª 91.0 ± 16.6b 92.0 ± 10.3b

0.0 ± 0.0a 0.0 ± 0.0a 2.0 ± 6.3a 0.0 ± 0.0a 2.0 ± 6.3a

96.0 ± 8.4b 100.0 ± 0.0b 100.0 ± 0.0b 100.0 ± 0.0b

0.0 ± 0.0a 0.0 ± 0.0a 0.0 ± 0.0a 18.3 ± 24.9ª


3.1 ± 4.7 7.7 ± 4.6bc 6.5 ± 7.5abc 15.2 ± 3.5cd

7.5 ± 10.4ab 7.0 ± 8.2ab 12.0 ± 10.3ac 6.0 ± 8.4ab 8.0 ± 7.9a


88.1 ± 11.9de 98.1 ± 3.6e 100.0 ± 0.0e 100.0 ± 0.0e

0.0 ± 0.0b 8.0 ± 10.3ab 11.0 ± 12.9ac 25.0 ± 17.2c

ab

Averages followed by equal letters in the same column do not differ statiscally at a significance level of 5%. Control = hydroalcoholic solution without thymol

Parasitol Res Table 3 Results of the sensory analysis, centrifugation and pH measurement of the emulsion and hydroalcoholic solution, at concentrations of 2.5 and 5.0 mg/mL, in the accelerated stability test. The pH values are the mean ± SD of three consecutive measurements of each sample. N normal, SM slightly modified, IM intensely modified Storage period and conditions

Emulsion

Hydroalcoholic solution

2.5 mg/mL

5.0 mg/mL

2.5 mg/mL

5.0 mg/mL

Sensory analysis pH measurement

N 6.78 ± 0.05

N –

N 6.12 ± 0.14

N 6.57 ± 0.03

Centrifugation

N

IM

N

N

Initial (24 h)

After 15 days, at room temperature (25 ± 2 °C), with light exposure Sensory analysis N

–

N

N

pH measurement

–

5.75 ± 0.07

6.16 ± 0.10

Centrifugation N After 15 days, at room temperature (25 ± 2 °C), without light exposure

–

N

N

Sensory analysis pH measurement

N 6.26 ± 0.04

– –

N 5.84 ± 0.08

N 5.86 ± 0.06

Centrifugation

N

–

N

N

After 15 days, in refrigerator (5 ± 2 °C) Sensory analysis pH measurement Centrifugation After 15 days, in oven (37 ± 2 °C)

6.35 ± 0.15

N

–

N

N

6.67 ± 0.21 N

– –

6.04 ± 0.11 N

5.96 ± 0.10 N

– – –

N 5.99 ± 0.21 N

N 5.91 ± 0.05 N

Sensory analysis N pH measurement 6.65 ± 0.05 Centrifugation SM After 15 days, under freeze-thaw cycles (40 ± 2 °C/4 ± 2 °C) Sensory analysis N

–

N

N

pH measurement Centrifugation

– –

5.80 ± 0.16 N

6.15 ± 0.03 N

6.46 ± 0.10 SM

1% (Daemon et al. 2009). For unengorged nymphs, both the emulsion and solution attained similar mortality rates to those obtained in a previous study using thymol dissolved in ethanol (Senra et al. 2013). Finally, in the case of the engorged nymphs, the groups treated with the emulsion suffered higher mortality at the concentrations below 5.0 mg/ml, and as of this concentration the mortality rates were similar to those of the previous study, where water + 1% DMSO was used. The hydroalcoholic solution, in turn, did not reach relevant mortality rates: they were below those reported by Monteiro et al. (2009). The variations observed among the different stages tested in this study can be attributed to the anatomic and physiological difference of Ixodidae (hard ticks) at each stage (larva/ nymph) and phase (engorged/unengorged), and the chemical properties of the formulation’s components. Like all arthropods, ticks are covered with a tegument, which besides body coverage and structural strength as the exoskeleton, provide protection against water loss and act as a source of reserve energy (Sonenshine 1991; Hackman and Filshie 1982). The cuticle is the outermost part of the

tegument. Although most of this structure defined by Hackman and Filshie (1982) as a Bheterogeneous, noncellular membrane^, is located on the external part of the tick body, there is evidence that it extends inside the body at determined points of communication with the tracheal system and ducts of the dermal glands. Its density is not uniform over the entire surface of the tick, so that there are thinner areas, mainly found at articulation points, and thicker and harder areas. The chemical composition of the cuticle is still not totally resolved, but it is widely assumed to be composed mainly of lipids, polyphenols, proteins, and chitin (Lees 1947; Hackman and Filshie 1982). It is believed that the waxes present in the outermost layer of the cuticle, called the epicuticle, are secreted by the dermal glands, which are drained by pores to the surface (Sonenshine 1991). This secretion occurs more intensely during and immediately after a blood meal or after exposure to light, heat, or mechanical irritation. This is more evident in nymphs and females (Lees 1947). Its function has been indicated as protection against desiccation (Sonenshine 1991).

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Most water loss of ticks occurs through the cuticle (Lees 1947), and the lipid layer present in this structure plays an important role in regulating this occurrence. This activity appears to function by means of a physiological control, as demonstrated by the regeneration of the lipid layer of the epicuticle of ticks after production of artificial injuries by abrasion (Lees 1947) and by changes in the proportion of lipids during the process of feeding and ecdysis (Hackman and Filshie 1982). It has been confirmed that various substances, such as detergents, when applied on the surface of the cuticle of ticks can increase the transpiration rate, leading to greater water loss (Lees 1947). Larvae and nymphs of Ixodidae have been found to be more susceptible to water loss than adults (Knüle and Rudolph 1982). Besides the water loss that occurs through the body tegument, additional water is lost by the gas exchange process during respiration (Sonenshine 1991). Adult hard ticks as well as nymphs have a respiratory system composed of spiracles and trachea. The larvae, however, do not have these structures and perform gas exchange exclusively through the cuticle (Hackman and Filshie 1982). Nymphs and adults have a valve mechanism that opens and closes the spiracles, acting as another physiological control against water loss (Needham and Teel 1991). The control of the opening and closing of the spiracles has been related to the water balance status of the animal, and according to Sonenshine (1991), engorged ticks are unable to keep the respiratory system closed, causing them to lose water faster. In this study, in both formulations, we used glycerin, a viscous substance with hygroscopic and moisturizing properties, for the purpose of keeping the formulation from drying out. This component forms a hydrophilic film on the surfaces where it is applied (Ribeiro 2006). The mortality rates found in the tests with unengorged larvae suggest a possible deleterious effect caused by glycerin associated with thymol, possibly resulting in increased water loss through the cuticle (Ravindran et al. 2011), or even the formation of a film causing occlusion of the channels responsible for gas exchange, since as mentioned above, at this stage, ticks do not have a tracheal respiratory system. This effect was not observed in the unengorged nymphs, and the mortality rates were similar to those recorded in a previous study, where a hydroalcoholic solution of 50% ethanol as the solvent was used (Senra et al. 2013). Since nymphs do not depend exclusively on the cuticle for gas exchange and absorption of water (because they have a tracheal system), the occlusive effects of the formulations on the tegument might have been counterbalanced by its respiratory activity, so that the deleterious effect of the adjuvants associated with thymol in the two formulations was attenuated. However, since no significant mortality occurred in the control groups, the mere obstructive action of glycerin does not explain the enhanced toxic effect of thymol. Thus, other pharmacological and/or physicochemical hypotheses should be investigated to clarify this enhancement.

The results of the tests with engorged larvae showed higher mortality rates in the groups treated with the emulsion. In the case of the hydroalcoholic solution, even at the highest concentrations tested, the mortality rate was not relevant. In a previous study investigating thymol dissolved in water +1% DMSO, relevant mortality rates (97%) were only attained starting at the concentration of 15 mg/mL (Daemon et al. 2009). The increased toxicity in the treatment with the emulsion on engorged larvae can be related to the use of adjuvants that promote permeation with lipophilic properties, such as cetearyl alcohol and grape seed oil. Given the liposolubility of thymol, we can suggest that the emulsion allowed stronger interaction of that substance with the cuticle’s fatty layer, facilitating its entry in the organism, and also keeping it in surface contact with the tick longer by the possibility of forming a film over the tegument. In the tests of the emulsion on the engorged nymphs, similar mortality rates were found to those reported in a previous study using an aqueous solution of 1% DMSO as solvent (Monteiro et al. 2009). On the other hand, lower mortality rates were noted in the tests with engorged nymphs and larvae treated with the hydroalcoholic solution. To understand this phenomenon, it is important to consider the higher production of waxes by the dermal glands that happens in the engorged phases, as explained previously. It is possible that the hydroalcoholic solution generated partial or inadequate solubilization of the waxes present on the cuticle’s surface, making penetration of the thymol less successful. On the other hand, the interaction of the lipophilic penetration agents with the thymol and the fatty layer of the cuticle can explain the fact the emulsion achieved satisfactory toxicity levels. With respect to the stability studies, the hydroalcoholic solution at concentrations of 2.5 and 5.0 mg/mL remained stable in all the storage conditions tested. The emulsion at 2.5 mg/mL, in turn, showed signs of instability (creaming) when subjected to high heat and freeze-thaw cycles. However, according to Brasil (2004), small alterations in these conditions are acceptable, frequent, and even expected. Besides this, there was no significant variation in the pH values which are suitable for application in the skin of dogs, considering that the average pH of the skin of a healthy dog varies between 5.86 and 6.45, with mean of 6.16 (Laurel, 2005). The emulsion at 5.0 mg/mL showed changes after the initial centrifuging, with signs of coalescence, so it was not tested under other conditions. Because this study is groundbreaking regarding the incorporation of thymol as an acaricide in formulations for topical use, the data presented here have considerable value as support for new studies. To better understand the action of the formulations tested here, it will be necessary to conduct further studies to determine the exact mechanism by which thymol acts, as well as the action of the moisturizing components used. Accompanying investigations to adjust proportions of the adjuvants, and possibly the addition of other excipients, can

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make the proposed formulations more stable, favoring a study in the near future of their acaricidal effect in vivo, thus, contributing to the validation of integrated strategies to control R. sanguineus sensu lato. Acknowledgements The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico [CNPq (National Council for Technological and Scientific Development)] and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior [CAPES (Coordination for the Improvement of Higher Education Personnel)], Brazil, for the financial support of this study.

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