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Evaluation of safety margins of Chenopodium album seed decoction: 14-day subacute toxicity and microbicidal activity studies

Background Sperm immobilizing activity and plausible mechanism of action of Chenopodium album seed decoction (CAD) have been elucidated in our earlier studies. The present study has been carried out to explore the safety standards of CAD along with microbicidal properties as prerequisite for its use as a topically applicable vaginal contraceptive. Methods The safety standards of CAD were assessed by a) Hemolytic index determination using rabbit erythrocytes, to set the doses of the other experiments, b) Dermal irritancy test using refined version of Draize scoring system on rabbits, c) Possible effect on local tissues and reproductive performance in female rats after fourteen daily single dose application, d) PCNA staining- to evaluate the effect of CAD on vaginal tissue proliferation, e) TUNEL assay- to examine its ability to induce in situ apoptosis in the vaginal tissue sections of the treated animals, and f) Microbicidal activity- to explore the effect of CAD on the growth of Lactobacillus acidophilus and Candida albicans. Results In vitro irritation studies on rabbit erythrocytes revealed the hemolytic index of CAD to be 8.2 mg/ml. The dermal irritation test showed it to be a non-irritant even at higher doses. Intra vaginal application of CAD in rat vagina for 14 consecutive days caused slight reversible inflammation on vaginal epithelial cells at doses as high as 82 mg/ml. However, at this dose level it neither had any adverse effect on vaginal tissue proliferation nor did it cause in situ apoptosis as evident from PCNA staining and TUNEL assay. Fertility and fecundity were restored 4-15 days after withdrawal of CAD application. At dose level 10 times that of its spermicidal MEC (minimum effective concentration), CAD did not block the growth of Lactobacillus, although the size of individual colony was marginally reduced. However, growth of the pathogenic fungus Candida albicans was completely inhibited with 20 mg/ml of CAD. Conclusion The overall result evolved from the study strengthens the candidature of CAD as a safe microbicidal spermicide. It is almost non-irritant to rabbit skin and rat vaginal tissues at doses 10 fold higher than its hemolytic index. The effect of CAD on Lactobacillus culture was not highly encouraging but it prevented the growth of the fungal pathogen Candida albicans at 20 mg/ml of CAD.


Background
The vaginal contraceptive products available so far are mostly based on Nonoxynol-9 (N-9), a mixture of oligomers [1]. As the healthcare industry moves towards using either pure compounds or mixtures whose individual components meet safety standards, the use of N-9 may or may not meet future safety regulations. Several European nations have already banned or restricted the use of N-9 and related detergent type spermicides, because repeated usage of these types of detergent type spermicides has been shown to cause vaginal/cervical irritation and even ulceration [2]. In addition, disturbance of the normal vaginal micro flora facilitates microbial infection and renders the subject susceptible to sexually transmitted diseases (STDs) [3]. Thus, development of safer non-detergent type spermicidal agent is the need of the day.
In our previous communications [4,5] we have discussed the spermicidal potential and plausible mechanism of action of Chenopodium album seed decoction (CAD). But unambiguous establishment of sperm killing activity is not enough to recommend it for clinical use unless its safety margin is ascertained in experimental animal models. It is well known that vaginal spermicides are products for topical application and are meant for use to prevent unintended pregnancies, besides offering protection against sexually transmitted diseases (STDs), including infection by human immunodeficiency virus (HIV). In general, topically applicable spermicidal molecules are exposed to the specific organs during their administration to the vaginal walls. Thus tolerance tests for specific organs prior to human exposure are very important. Moreover, recent studies on currently available vaginal spermicides have revealed that they do not actually provide protection against STDs; rather long term use of such detergent type spermicides is associated with vaginal ulceration, which facilitates the entry of foreign pathogens [6]. Keeping in view the toxic effects exerted by the existing spermicidal agents, we carried out a series of safety measurement studies with CAD in experimental animals.
Mammalian vaginal bacterial flora comprising of Acidophilus play a major role in maintaining vaginal health by prohibiting growth of other pathogenic microbes and STDs [7]. No spermicidal preparation is acceptable if it has any deleterious effect on the normal vaginal flora. Taking this fact into consideration we studied the effect of CAD on the growth of Lactobacillus acidophilus a normal habitat of women vagina. On the other hand, Candida albicans is the most commonly encountered fungal pathogen of the human vagina [8]. We therefore took this strain as representative pathogenic strain and explored the effect of CAD on its growth. Moreover, considering the detrimental effect of CAD on sperm DNA [5], it was necessary to examine whether continuous application of CAD may disturb the physiological processes that normally occur during the estrous cycle such as proliferation and apoptosis.

Animals
Toxicology studies were conducted in both rodent (viz. rat) and non-rodent (viz. rabbit) species. Animals were maintained under light and temperature control (complying with standard husbandry conditions) with food and water adlibitum and were acquired from our institute's animal facility. All experiments were performed in accordance with the guidelines formulated by the animal ethics committee of Indian Institute of Chemical Biology.

Experiment-1
Determination of hemolytic index for ascertaining the in vitro cytotoxic dose Haemolytic index, i.e. the concentration required for 50% hemolysis of [9] RBCs, was ascertained for CAD to determine the subsequent doses to be used for topical application in sub acute toxicity studies. Freshly isolated rabbit blood in isotonic PBS (35 mM phosphate buffer, 150 mM NaCl, pH 7.0) was initially centrifuged at 2000 g for 10 minutes. RBCs were washed (3 x) and resuspended (130 × 10 6 cells/ml) in PBS, pH 7.4. Test samples were prepared by combining 0.5 ml of the RBC suspension with 3 ml of PBS containing 2 mg/ml, 4 mg/ml, 8 mg/ml and 10 mg/mlof CAD for 30 min. Negative controls were prepared by replacing the CAD solution by PBS. Positive controls were prepared by incubating the cells with 1% Triton-X 100. In addition, positive control samples underwent sonication and mild shaking to ensure complete hemolysis of the sample. All samples were run in triplicate. The experimental samples were centrifuged (1000 g) for 3 min and placed in ice bath to quench the hemolytic reaction. Following the quenching of the hemolytic process, the absorbance of the supernatant of each sample was determined at 576 nm. The percentage of hemolysis was calculated on the basis of the following equation. where Abs is the absorbance of the sample, Abs control is the absorbance of the control sample (negative control), and Abs 100 is the absorbance of the sample in which 100% hemolysis occurred (positive control).

Experiment-2
Safety studies in experimental animal model Low volume eye test (LVET) Low volume eye test (LVET) [10] is the refined version of Draize eye irritancy test [11] that uses 1/10 th volume of the test substance placed directly on the cornea. The level and duration of eye irritation in the LVET are less than those in the Draize procedure, which means that it is a less stressful test. Moreover LVETs are more predictive of human response.
In this test one eye served as the target and the other as control. On the day of dosing, the test material (0.1 ml of 8.2 mg/ml or 82 mg/ml of CAD in physiological saline) was applied into the conjunctival sac of one eye of three rabbits. The lower eyelid was held open momentarily during dosing. The eyelids were then gently held together for a moment and then released to allow the animal to blink freely. The other eye of each rabbit was treated similarly with normal saline and served as a control. The cornea, iris, and conjunctiva of the treated and control eyes were examined at 1, 24, 48, and 72 hr after treatment. The degree of eye irritation was evaluated following the criteria of Draize et al [11].

Dermal irritation test
Since a clinical formulation is yet to be prepared, a galenic formulation of CAD prepared with K-Y Jelly (Johnson and Johnson India Ltd., Mumbai, India) at hemolytic dose (8.2 mg/ml), at a dose (82 mg/ml) 10 fold higher than the hemolytic index, and saline (placebo) were applied topically (0.5 ml per site, applied uniformly on a 2.5 cm × 2.5 cm area) to one intact and one abraded test sites per rabbit. The test sites were occluded by covering each site with a dressing gauze pad (3 × 3 cm) and over wrapping the site with a perforated nonirritating plastic tape (100 μm) for 24 hr following application of the gel. Dermal irritation was measured following the index of Draize scoring system at 24, 48 and 72 hour after gel application [11].

Experiment-3
Test for possible effect and reproductive performance in female rats after fourteen daily single dose applications Twelve female Sprague-Dawley rats having regular fourday estrous cycle were used in this evaluation. The animals were divided into four groups each consisting of three rats. K-Y jelly based CAD, at doses 8.2 mg/ml and 82 mg/ml, was administered intra vaginally in each animal (100 μl) of two groups for 14 consecutive days. The formulations were placed deep into the vagina through a syringe. The first group (Group I) was left untreated, the second group (Group II) was treated with only K-Yjelly (the placebo), the third group (Group III) received a dose of 8.2 mg/ml (hemolytic index), and the fourth group (Group IV) received a dose of 82 mg/ml (10× hemolytic index). Rats were individually observed daily for overt clinical signs (vaginal swelling, redness, and discharge including bleeding). The change in the regularity of estrous cycle was observed by microscopical examination of vaginal lavage. Each of the female rats in their proestrous phase was placed after completion of the treatment in the cage of a proven male and next day its vaginal smear was examined. Presence of sperm in the lavage was considered successful mating and day-1 of pregnancy. The mated females were allowed to complete their term and the litter size and body weights of the pups were recorded for comparison with the control group. The pups were maintained until weaning and their general growth rates were compared with those of pups from the control group.

Experiment-4
Effect of 14-day (sub acute) vaginal administration of high doses of CAD on local tissues, vaginal tissue proliferation, and in situ apoptosis Regularly cycling 12 adult Sprague Dawley rats were divided into four groups (control and treated). Animals of Group-I served as untreated control and those of Group-II were treated with placebo gel (K-Y jelly only). Animals of Group-III and Group-IV were treated vaginally with 8.2 mg/ml and 82 mg/ml of CAD solution in K-Y jelly respectively. All animals were weighed on the first day of dosing and every seventh day thereafter, and were sacrificed at the end of the treatment schedule. The vaginal tissues were slit open ventrally between the urethral orifice and fornix. Representative samples of the proximal, middle and distal portions were collected and fixed in 10% neutral buffered formalin fixative. Fixed tissues were embedded in paraffin, sectioned at thickness of 5 μm, stained with hematoxylin and eosin, and examined under × 200 magnification using a Leica light microscope (Milton Keynes, Buckinghamshire, UK) interfaced with an image analysis system. The images were captured using the image pro plus program in conjunction with a 3CCD camera and images were transferred to Adobe Photoshop 7.0 software for observation and analysis. The vaginal tissue sections were observed for epithelial ulceration, edema, leukocyte infiltration, and vascular congestion.

Cell proliferation (PCNA staining)
The expression level of Proliferating Cell Nuclear Antigen (PCNA) has broad correlation with mitotic activities and therefore can be used as a marker for cell proliferation. Here PCNA staining was performed to evaluate the cell-proliferating activity of CAD treated vaginal tissues. Immunohistochemistry of tissue sections was performed using Zymed PCNA kit (South San Francisco, CA). Deparaffinised tissue sections were treated to remove the endogenous peroxidase activity, and then incubated with a biotinylated anti-PCNA monoclonal antibody. Sections were washed in phosphate buffered saline (PBS), incubated with streptavidin-peroxidase for 30 minute at room temperature, and washed with PBS. Positive control consisted sections of mouse intestine known to express PCNA and negative control were prepared with the sections stained without the primary antibody. The bound horseradish peroxidase complexes were developed using diaminobenzidine tetrahydrochloride according to the manufacture's instructions. The sections were counterstained with Harris' hematoxylin, dehydrated, mounted with glass cover slips, and were then observed under a Leica light microscope (Milton Keynes, Buckinghamshire, UK). The nature of staining and the distribution of PCNA reactivity were determined by scoring a minimum of 100 cells in the vaginal tissue sections in several random fields and percentage of PCNA positive cells were calculated in each field.
In situ apoptosis (TUNEL assay) The ability of CAD to induce in situ apoptosis in rat vaginal tissue was evaluated by the terminal deoxynucleotidyl transferase (TdT)-mediated digoxigenin-uridine triphosphate (dUTP) nick-end labeling (TUNEL) assay, which labels the exposed 3 ' hydroxyl ends of fragmentated nuclear DNA [12]. The assay was performed by using the DNA fragmentation kit obtained from Clontech Laboratories Inc. (Mountain view, CA, USA). This kit uses a direct labeling procedure for detecting DNA fragmentation at the single-cell level. Control and CAD treated tissue sections were deparaffinised and rehydrated in graded alcohol. The tissue sections were then treated with proteinase k (20 μg/ml) for 15 min at room temperature to digest nuclear matrix protein and expose the chromatin. Sections were then incubated for 1 hr with TdT and FITC-dUTP in a humidified chamber at 37°C. After terminating the reaction, sections were washed in PBS and nuclear staining was performed by counterstaining the sections with propidium iodide (PI, 1 μg/ml). Positive control slides were prepared by treating the control sections with DNase (2 μg/ml) in reaction buffer [13] for 10 minute prior to TUNEL staining. Negative controls were prepared by omitting TdT from the reaction mixture. These were then observed under an Olympus Bx51 fluorescence microscope (Olympus Corporation, Lake Success, NY) and the TUNEL positive cells were counted in several random fields per tissue section (x200) and the percentage of TUNEL positive cells were determined.

Experiment-5
Tests for microbicidal activity of CAD Effect of CAD on Lactobacillus acidophilus The media for culture of bacteria were purchased from M/s Hi-Media Laboratories Pvt. Limited, Mumbai, India. Spores of Lactobacillus acidophilus were obtained from pharmaceutical capsules marketed by Infar (India) Limited. To obtain direct counts of lactobacilli, sterile, molten (45-50°C) Lactobacilli MRS agar was poured into sterile Petri dishes with CAD or without CAD. Test plates in triplicate containing 2 mg/ml(at MEC), 10 mg/ml(5 × MEC) and 20 mg/ml(10 × MEC) of CAD were inoculated with spores of Lactobacillus acidophilus (~50 spores/90 mm plate), and incubated at 37°C in an atmosphere containing 5% CO 2 and 95% air for a period of 72 hr. Control plates were inoculated simultaneously and incubated in a similar manner. The number of colonies and the size were determined at the end of experiment.

Effect of CAD on fungal growth
The species of fungi used in the present study was Candida albicans. Sterile molten potato dextrose agar (PDA) was poured into sterile Petri dishes (a) with CAD and (b) without test reagents (as control). The test plates in triplicate containing 2, 10 and 20 mg/ml CAD were inoculated with C. albicans spores with a sterile inoculating loop and incubated at 25-30°C in an inverted position keeping the agar side up with increased humidity. Control plates were incubated in similar manner for 24 hr. The number of colonies and their size were measured at the end of the experiment.

Statistical analysis
Data are represented as mean ± SEM and significance was calculated from Student's t test. P < .05 was considered as the criterion for statistical significance.

Experiment 1
The percentage of hemolysis was plotted against concentrations of CAD and the hemolytic index was determined from the curve. The results of hemolytic index studies indicated that concentration required for 50% hemolysis was 8.2 mg/ml, which is four fold higher than its spermicidal MEC of CAD ( Figure 1).

Experiment-2 Safety studies in experimental animal model
Low volume eye test (LVET) The rabbits closed their eyes immediately after treatment and there was no significant lapse of time between opening of the two eyes receiving either CAD or the vehicle; however; the groups treated with 8.2 mg/ml and 82 mg/ml CAD solutions, exhibited mild blinking. The eyes became normal after 7(± 1) min. At 1 h post treatment the eyes were fully open; the pupil was smaller in the treated eyes than in the control eye, but regained normal size by the next two hours. Dermal irritation test There was no persistent erythema or oedema in any of the animals. The irritation indices as recorded in different treatment groups after 24, 48 and 72 hours of treatment are given in the Table 1.

Experiment-3
Test for possible effect on reproductive performance in female rats after fourteen daily single dose application Rats of group I and II were found to be normal and similar in all aspects. The animals of group III showed an average prolongation of diestrous phase by one to two days, but in group IV the vaginal smear showed persistent diestrous phase from the day after application. The first proestrous phase appeared in group III (8.2 mg/ml) after 4(± 2) days of cessation of treatment. In Group IV (82 mg/ml), the animals had their first proestrous phase restored 15(± 2) days after discontinuation of treatment. All the treated animals which regained their normal proestrous vaginal smear mated successfully and completed their pregnancy. The litter size from their delivery was normal and the pups were apparently normal. The results suggest that CAD does not affect female fertility.

Experiment-4 Effect of 14-day (sub acute) vaginal administration of high doses of CAD on local tissues, vaginal tissue proliferation and in situ apoptosis
Effect on local tissues Intra vaginal administration of CAD at doses up to 82 mg/ml for 14 consecutive days did not cause any inflammatory reaction in the vaginal epithelium. Figure 2 shows the representative vaginal sections of rats. Light microscopic examinations revealed intact vaginal tissues of rat following daily intra vaginal administration of K-Y jelly based placebo alone (Figure 2a) or K-Y jelly based CAD at doses up to 82 mg/ml (Figure 2b). There were slight inflammatory changes. PCNA staining Immunohistochemical detection of PCNA was used to visualize the proliferative activity in paraffin sections of control (placebo) and CAD treated vaginal tissues. PCNA positive cells identified as dark stained nuclei (Figure 3) were present in both control and CAD treated vaginal epithelium. Table 2 summarizes the percentage of PCNA positive cells in control and CAD treated vaginal tissues. More than 60% of vaginal epithelial cells in the placebo group expressed PCNA and no significant difference in mean percentage of cells expressing PCNA was observed in tissues treated with 82 mg/ml CAD, which implies that intra vaginal administration of CAD did not cause significant reduction in the number of cycling cells in the vaginal epithelium. In situ apoptosis by TUNEL assay TUNEL assay followed by fluorescence microscopy revealed that repeated vaginal administration of CAD at dose 82 mg/ml did not cause any significant difference in apoptosis in the female vaginal epithelium as compared to the placebo group. Only minimal apoptotic cells were detected in both groups of tissues ( Figure 4). There was no significant difference in the mean percentage of TUNEL positive cells among those exposed to K-Y jelly alone (14 ± 0.42%) or to gel containing 82 mg/ml (17 ± 0.71) of CAD when counted in a microscopic field.

Experiment-5
Tests for microbicidal activity of CAD Effect of CAD on Lactobacillus acidophilus Comparative growth of L. acidophilus in agar plate is depicted in Figure 5. The results showed that CAD did not affect the number of colonies (control: 30 ± 0.521 and CAD 20 mg/ml: 28 ± 0.421) of Lactobacillus in laboratory culture at least up to a concentration of 20 mg/ml. However, there was a significant reduction in individual colony size, which implies that CAD might have some adverse effect on Lactobacillus growth. Effect of CAD on fungal growth There was no fungal growth in presence of CAD at concentration 20 mg/ml as depicted in Figure 6. At this dose level, CAD strictly inhibited the growth of C. albicans.

Discussion
To be an ideal spermicidal contraceptive, the candidate spermicide should fulfill the following criteria: (i) longterm application of the spermicide should not bring local organic irritation, (ii) it should not alter the microbial balance in the female genital tract which may facilitate the entry of foreign pathogens, and (iii) repeated application of the spermicide should not have any adverse effect on reproductive performances and subsequent fertility of the female rats. Considering these points, the toxicity level of CAD was evaluated in experimental rat and rabbit models. The hemolytic index is a rapid screening assay of first order for the assessment of acute irritation potential of topically applicable microbicide or spermicide [9]. It helps in the determination of subsequent doses to be used for toxicity studies. The hemolytic index of CAD was found to be 8.2 mg/ml, which is four fold higher of spermicidal MEC. A jell type formulation was made with K-Y jelly (a non toxic, inert lubricant jelly which is widely used in human as well as animals) in doses of CAD ranging up to 10 folds of the hemolytic index (82 mg/ml) was used for local tolerance tests.
The results of 14-day sub acute toxicity studies indicated that the vaginal tissue irritation caused by CAD was insignificant at doses as high as 82 mg/ml of CAD and no adverse effect on subsequent fertility was observed after repeated intra vaginal application of CAD. On cessation of treatment, when the animals were allowed to mate, they conceived and delivered normal off springs indicating that genital tract was not affected despite repeated exposure to high doses of CAD for 14 consecutive days but the regularity in the estrus cycle of the treated animals (at dose 82 mg/ml) were affected may be due to some changes in the submucosal microenvironment caused by high dose of CAD application. Further studies are required to explore the effect of CAD on vaginal cytology. The results of PCNA staining ( Figure 3) and TUNEL assay ( Figure 4) showed that CAD neither hindered the normal cell proliferation of the vaginal tissues nor caused apoptosis in the rat vaginal cells. This suggested that the spermicidal activity and ROS mediated DNA damaging potential of CAD is a cell specific phenomenon rather than a general event a b  [3,4]. Currently used spermicides are mainly N-9 based. There is a growing concern that chemical irritations brought by the long term use of such spermicides disrupt the vaginal mucosa and might actually increase the risk of STDs including HIV transmission in sexually active women [2]. The studies on microbicidal activities of CAD showed that CAD prevents the growth of the most commonly encountered fungal pathogen of human vagina, Candida albicans; but the effect of CAD on Lactobacillus culture was a bit different. CAD did not prevent the growth of this normal vaginal habitat (Figure 5) but there was reduction in the individual colony size in presence of CAD. This implies that CAD might have some adverse effect on its growth which requires further investigation. The major constituent of CAD is a mixture of triterpene glycosides [4] of amphiphilic nature that is likely to undergo aggregation in solution, and this might contribute to its irritation potential and microbicidal effects. Earlier work has demonstrated that co-precipitation of these amphiphilic compounds with water-soluble polymers like polyvinylpyrrolidone decreases their self associating properties of leading to a decrease in irritation potential without compromising spermicidal activity [14]. Further investigation in this direction may be undertaken to reduce the adverse impact of CAD on Lactobacillus.
Use of a vaginal product, for contraceptive purpose, may be a life-long practice and as a consequence long term effect of the product should be considered in a form of   repetitive drug administration. Therefore, chronic dosing in animals is needed to identify potential problems with long-term product use. Either one-year studies in two animal species (one rodent and one non-rodent), or two year rodent carcinogenicity bioassays, should be conducted. It is worthy to mention that the ultimate goal of the study is for the beneficial of human race and thus the effect of CAD on human sperm along with the referred studies have to be considered. The observations made in this paper, may therefore be viewed as a start in evaluating the toxicity of CAD for contraceptive goals.

Conclusions
In summary, our present investigation reveals that the spermicidal effect of CAD is not a non specific surfactant type action on sperm; rather it is a cell specific phenomenon. Local toxicity studies on CAD revealed that its 14-day daily intra vaginal administration at high doses neither prevented vaginal tissue proliferation nor it caused apoptosis to the vaginal tissues and the fertility potential of the treated animals were restored immediately after withdrawal of CAD treatment.