The study was approved by The Investigational Review Board Inc. (IRB # Lf 001-0011) and was conducted in accordance with Good Clinical Practices and with the principles outlined in the Declaration of Helsinki. The study was registered with ClinicalTrials.gov Protocol Registration System ID number NCT01514513 and all study treatments with follow up assessments were performed by the authors listed residing at South Florida Family Health Research Center, 6971 West Sunrise Blvd., Suite 102, Plantation, FL 33,313.

Individuals, aged 4 years or older, having a single place of residence, diagnosed with an active head lice infestation of at least 10 live lice at the Screening visit and a presence of nits were eligible for inclusion in this study. The subjects agreed to not use a lice comb, or any other pediculicides or medicated hair grooming products for the duration of the study (through Day 15 visit). Furthermore, parents and other family members of a child were also screened for head lice. If other household members were found to have head lice and were eligible, they were either enrolled in the study or were treated with the same product and in the same manner as study participants. All the participants of the study and where appropriate their legal guardian were explained the study’s procedures and signed informed consents.

Subjects using any form of head lice treatment for at least four weeks or any topical medication for a period of 48 hours prior to the Screening visit (Day 1) were not eligible for inclusion in the study. Individuals were also not eligible for participation if taking systemic or topical medications (including antibiotics), or suffering from visible skin/scalp condition at the treatment sites, of which in the opinion of the investigative personnel would interfere with the evaluation of the test products. Anyone who was allergic or sensitive to ragweed or any ingredient in either test product, pregnant or nursing, who did not understand the subject requirements for study participation and/or exhibited poor compliance with the required visits were also excluded from the study.

Subjects could withdraw from study treatment at any time at their own request, or at the discretion of the investigators for safety, behavioral or administrative reasons.

Subjects were randomly assigned by sealed-envelop randomization to receive Sodium Chloride or Permethrin during the treatment period. An equal number of subjects were allocated into each arm of the study. This was an open-label study; as such, the investigators, site personnel and the subjects were aware of the product being used.

Test product was 1% Sodium Chloride (0.1709 M)

spray (LiceFreee Spray^®, in 6 ounce bottles; Lot number J1821 by Tec Laboratories, Inc. Albany, Oregon), and reference product was 1% Permethrin Crème Rinse (Nix^®, Insight Pharmaceuticals, LangHorne, PA, in 2 fluid ounce bottles, 280 mg/fluid ounce; Lot number NX0908—Expiration 3/2012). The doses used for test and reference products were strictly followed as outlined on product label directions.

Subjects had the products applied on Day 1 by investigative site personnel after determination of meeting study inclusion criteria. The time of application, the weight of the bottle before and after application, the time elapsed until the hair was considered dry (for Sodium Chloride spray), the start and end time of rinsing (for Permethrin) were recorded.

In the Sodium Chloride spray arm, the product was sprayed on the hair until the hair was completely saturated. The hair was then allowed to dry naturally. Efficacy evaluation was performed 1 hour after the application of Sodium Chloride or earlier if the hair was completely dry using a metal comb. The participants were asked to wait 24 hours before returning to their normal hair hygiene. For those in the Permethrin Crème Rinse treatment group, the hair was washed with baby shampoo (Johnson and Johnson, New Brunswick, NJ) without conditioner and then rinsed with water. The hair was towel dried. Permethrin Crème Rinse was then applied to saturate the hair, scalp, behind the ears and to the nape of the neck. After 10 minutes of treatment with Permethrin Crème Rinse, the hair was rinsed with warm water and towel dried and efficacy of the treatment was assessed using a metal comb. Subjects received treatment at the time they were in the clinic which varied based on appointment time. No nit combs were used during the study. If live lice were observed at the second visit (Day 8), the subjects received a second treatment with the assigned test products following the same procedures mentioned above.

After the initial application of the products on Day 1, evaluation and follow-up visits were performed at day 7 and day 14, respectively, with a ±1 day window (Day 8 and Day 15 visit). Demographic parameters such as date of birth, gender, race, ethnicity, size of household, and hair characteristics (length, texture, and curliness) were recorded together with medical history and current medication usage.

During examination, hair and scalp were assessed for the number of live lice at pre-treatment, immediately after the treatment at Day 1 (and if applicable, on Day 8)at Days 8 and 15. The detection of any live lice on a subject was considered a treatment failure with either product used. Numbers of live lice and nits observed in the left, middle, and right side of the head were recorded for all time points but the number of nits was estimated only prior to the course of treatment.

Assessment of other symptoms such as pruritus, erythema, presence of secondary infection, and excoriation was recorded at each visit using a 4-point scale (none 0, mild 1, moderate 2, or severe 3). Vital sign measurements such as systolic, diastolic blood pressure and pulse rate, examined after the subject rested in a seated position for at least 5 minutes, and evaluation of existence of red eyes were performed at each visit. Urine pregnancy test was performed at the Screening Visit (Day 1) only to ensure all females of child-bearing potential were not pregnant.

The primary measurements were the safety and efficacy in the reduction of the total number of lice observed on the subject’s head and scalp after treatment assessed on Day 1, 8 and 15. An evaluation was performed at each subject’s visit to determine whether their eyes were clear (value recorded 0) or red (value recorded 1). Additional assessments during the study for frequency of pruritus, erythema, presence of secondary infection, or excoriation were performed at each subject’s visit using a four point scale detailed above [16-18].

The sample size of 20 subjects per treatment group was a feasible sample size to provide descriptive statistics for the efficacy and safety parameters as no formal calculation of power or sample size was performed in this study. Fisher test and t-test were used when applicable to compare the efficacy of 1% Sodium Chloride spray product in eliminating head lice to that of Permethrin.

The following parameters were calculated: The proportion of subjects that were 100% free of live lice in each treatment group immediately post-dosing, and at the Day 8 and Day 15 visits and the proportion of subjects that required retreatment to be 100% free of lice at day 15 visit was recorded. For comparison, Fisher test and two sided 95% confidence interval were performed on the difference in proportion between treatment groups. The numbers of live lice per head before the treatment and after each visit were also counted for the comparison between treatments. A p-value < 0.05 was considered statistically significant.

Forty-two subjects participated in the study were assigned equally into each treatment group (Sodium Chloride spray or Permethrin). All females of child-bearing potential had negative pregnancy tests on Day 1 prior to their participation in this study. One subject in each treatment group completed the first treatment and first assessments but dropped out of the study prior to Day 8, and twenty subjects in each treatment group completed the study.

As shown in Table 1, there were no differences in age, race, and gender in the two treatment groups. Nearly half of all subjects had average texture to the hair (47.6% in both treatment groups) but the curliness and length varied. The number of individuals with long and extra-long hair was higher in Sodium Chloride spray arm while curly hair occurred more in Permethrin arm.

Table 1. Each treatment group’s subject demographic characteristics.

Immediately following treatment assessment (Figure 1) on Day 1, 79.2% Sodium Chloride Spray subjects (16 of 21) and 66.7% Permethrin subjects (14 of 21) were lice free (p > 0.05). At Day 8 visit, 25.0% subjects (5 out of 20) in Sodium Chloride Spray arm and 75.0% subjects (15 out of 20) in Permethrin arm required another course of treatment (p < 0.05). Of those requiring retreatment at Day 8 visit, 3 of 5 Sodium Chloride spray subjects (60.0%) were successfully treated as compared to 5 of 15 Permethrin subjects (33.0%) (p > 0.05). Figure 2 presents the percentage of subjects free of lice at the end of the course of treatment (Day 15 visit), 85.0% of the subjects in Sodium Chloride Spray arm and 45.0% of the subjects in Permethrin arm (p < 0.05).

The number of live lice presented prior to the initial treatment was comparable in both treatment groups (21.76 range 10 to 68 lice and 21.29 range 10 to 60, p > 0.05, for Sodium Chloride Spray and Permethrin Crème Rinse, respectively). Immediately after treatment on Day 1, treatment failure subjects treated with Sodium Chloride Spray had 1.95 ± 1.28 lice as compared to 2.95 ± 6.27 in the Permethrin group (p > 0.05). At Day 8, the number of lice in subjects of the Sodium Chloride Spray treatment failure group was 1.40 ± 2.19 as compared to 2.95 ± 2.98 in the Permethrin group (p > 0.05).

At Day 15, the number of lice observed in the treatment failure subjects of the Sodium Chloride Spray group was 0.55 ± 1.5 compared to 5.45 ± 7.91 in the Permethrin group (p < 0.01). The summary of the efficacy results is presented in Table 2.

Sixteen of the twenty-one subjects in the Sodium Chloride Spray arm received only the first application on day one (all were free of lice on day eight assessment) while another 6 subjects received a second application on day eight. One subject failed to return for the day eight assessment. This compares to six subjects receiving one application (subjects were free of lice on day eight assessment) and another 15 subjects receiving a second application of 1% Permethrin Crème Rinse on day eight. Again one subject failed to return for the day eight assessment.

As presented in Table 3, the secondary characteristics such as pruritus, erythema, secondary infection, and excoriation of the lice infestation from Sodium Chloride Spray treated group were comparable to the control group. However, the improvement in pruritus in subjects treated with Sodium Chloride was consistently greater compared to Permethrin Crème Rinse (day one 1.14 + 1.1 versus 0.71 + 0.49; day 15 0.29 + 0.47 versus 0.43 + 0.53 for Sodium Chloride and Permethrin, respectively) however, these values were not statistically significantly different due to the number of subjects included in the study. One subject (white, Hispanic, 11 year old female with extra-long, fine, straight hair), with Sodium Chloride spray, reported a headache on Day 4 of the study, for which she self-administered one dose of ibuprofen (Motrin^®). This adverse event was the only one reported in the study and she was a treatment failure.

No abnormal or significant differences in vital signs were measured for subjects in this study between the treatment groups. During the study, no serious adverse events were observed. Future studies are needed to fully assess adverse events as the number of subjects in the study was low.