Preparation and Characterization of an Oral Norethindrone Sustained Release/Controlled Release Nanoparticles Formulation Based on Chitosan.

Norethindrone has short half-life and low bioavailability. The objective was to prepare an oral Sustained Release/Controlled Release (SR/CR) Liquid Medicated Formulation (LMF) to enhance bioavailability and improve patient compliance. Norethindrone was solubilized in HP-ß-CD then complexed with different concentrations of Low Molecular Weight Chitosan (LMWC) (mucoadhesive). PolyElectrolyte Complexes (PECs) were homogenized with oleic acid using different concentrations of tween 80 to form LMFs (nanoemulsions). PECs and LMFs were characterized using different techniques. LMF 2 (optimum formula containing 2.5% w/v LMWC 11 kDa) was administered orally to dogs and mice for pharmacokinetic and adhesion evaluation. DSC, FTIR spectroscopy and SEM images indicated complex formation. Mean diameters of PECs were 183-425 nm, mean zeta potentials were + 18.6-+ 31 mV, and complexation efficiencies were 18.0-20.6%. Ten to fifteen percent tween was needed to prepare homogenous LMFs. Mean diameter of LMF 2 was 10.5 ± 0.57 nm, mean zeta potential was - 11.07 ± - 0.49 mV, encapsulation efficiency was 95.28 ± 1.75%, and each mL contained 145.5 µg norethindrone. SEM images showed spherical homogeneous oil droplets. All of these parameters were affected by molecular weight and concentration of chitosan. Norethindrone release from LMFs was controlled (zero order) for 96 h. It was little affected by molecular weight and concentration of chitosan but affected by concentration of tween 80. LMF 2 adhered to GIT for 48 h and enhanced the bioavailability. It showed no cytotoxicity after considering dilution in GIT and was stable for 3 months refrigerated. In conclusion an effective SR/CR LMF was prepared.

Metabolite Identification, Reaction Phenotyping, and Retrospective Drug-Drug Interaction Predictions of 17-Deacetylnorgestimate, the Active Component of the Oral Contraceptive Norgestimate.

Ortho Tri-Cyclen, a two-drug cocktail comprised of ethinylestradiol and norgestimate (13-ethyl-17-acetoxy-18, 19-dinor-17α-pregn-4-en-20yn-3 oxime), is commonly prescribed to avert unwanted pregnancies in women of reproductive age. In vivo, norgestimate undergoes extensive and rapid deacetylation to produce 17-deacetylnorgestimate (NGMN), an active circulating metabolite that likely contributes significantly to norgestimate efficacy. Despite being of primary significance, the metabolism and reaction phenotyping of NGMN have not been previously reported. Hence, detailed biotransformation and reaction phenotyping studies of NGMN with recombinant cytochrome P450 (P450), recombinant uridine 5'-diphospho-glucuronosyltransferases, and human liver microsomes in the presence and absence of selective P450 inhibitors were conducted. It was found that CYP3A4 plays a key role in NGMN metabolism with a fraction metabolized (fm) of 0.57. CYP2B6 and to an even lesser extent CYP2C9 were also observed to catalyze NGMN metabolism. Using this CYP3A4 fm value, the predicted plasma concentration versus time area under the curve (AUC) change in NGMN using a basic/mechanistic static model was found to be within 1.3-fold of the reported NGMN AUC changes for four modulators of CYP3A4. In addition to NGMN, we have also elucidated the biotransformation of norgestrel (NG), a downstream norgestimate and NGMN metabolite, and found that CYP3A4 and UGT1A1 have a major contribution to the elimination of NG with a combined fm value of 1. The data presented in this paper will lead to better understanding and management of NGMN-based drug-drug interactions when norgestimate is coadministered with CYP3A4 modulators.

Degradation of gestodene (GES)-17α-ethinylestradiol (EE2) mixture by electrochemical oxidation.

Evidence of the negative effects of several pharmaceutical molecules, such as hormones and steroids, on the environment can be observed throughout the world. This paper presents the results of the anodic oxidation of the mixture of gestodene steroid hormones and 17 α-ethinylestradiol present in aqueous medium. The tests were conducted in an undivided cell containing a working volume of 50 mL, using a Na2SO4 solution as support electrolyte and boron-doped diamond electrodes. The experiments were adjusted to the structure of a 33 factorial design. The evaluated factors were: support electrolyte concentration (0.02, 0.05, and 0.10 M), pH of the reaction media (2, 3, and 4), and current density (16, 32, and 48 mA cm-2). Under the optimum conditions (0.02 M Na2SO4, pH 4, and current density of 32 mA cm-2), the degradation of at least 93% of the initial concentration of gestodene and 17α-ethinylestradiol was reached in a reaction time of 5 and 10 min, respectively. The complete degradation of both molecules required 15 min of reaction. Under these conditions, the degradation profile of the pharmaceutical mixture as each one of the active ingredients, followed a pseudo-first order kinetic behavior (kmix = 0.0321, kGES = 0.4206, and kEE2 = 0.3209 min-1).

Preparation and in vitro/in vivo evaluation of gestodene (GEST) intravaginal ring.

Preparation and in vitro/in vivo evaluation of gestodene (GEST) intravaginal ring (IVR) formulations which can release a constant dose of GEST during 3 weeks were investigated. In present study a reservoir gestodene intravaginal ring, including a gestodene silicone elastomer core and the non-active silicone layer, was reported, which was manufactured by reaction injection moulding at 80°C for 20 min. The raw materials compatibility experiments showed that the silicone elastomer core carrier wouldn't interact with drugs. In vitro release samples were determined by HPLC and the experiment was performed under sink conditions. The equation of cumulative release verse time was Y=64.76χ+5.44 (r=0.9998), performing zero-order release at about the target dose of 60 µg/day over 21 days. Drug release increased with temperature elevating from 45 to 55°C, which could be attributed to optimizing the prescription. In addition, the pharmacokinetic and safety studies of gestodene intravaginal ring were evaluated in female New Zealand White rabbits. The GEST in plasma was analyzed by LC-MS/MS and the results proved that the correlation between in vitro and in vivo was relatively well.

Investigation on the Interaction of Norgestrel with Human Serum Albumin Using Spectroscopy and Molecular-Docking Method.

The interaction of norgestrel with human serum albumin (HSA) was investigated by spectroscopy and molecular-docking methods. Results of spectroscopy methods suggested that the quenching mechanism of norgestrel on HSA was static quenching and that the quenching process was spontaneous. Negative values of thermodynamic parameters (ΔG, ΔH, and ΔS) indicated that hydrogen bonding and van der Waals forces dominated the binding between norgestrel and HSA. Three-dimensional fluorescence spectrum and circular dichroism spectrum showed that the HSA structure was slightly changed by norgestrel. Norgestrel mainly bound with Sudlow site I based on a probe study, as confirmed by molecular-docking results. Competition among similar structures indicated that ethisterone and norethisterone affected the binding of norgestrel with HSA. CH3 in R1 had little effect on norgestrel binding with HSA. The surface hydrophobicity properties of HSA, investigated using 8-anilino-1-naphthalenesulfonic acid, was changed with norgestrel addition.

A randomised study comparing the effect on ovarian activity of a progestogen-only pill (POP) containing desogestrel and a new POP containing drospirenone in a 24/4 regimen.

OBJECTIVES: Progestogen-only pills (POPs) are safer with respect to cardiovascular risks than contraceptives containing estrogens. Despite the increased contraceptive efficacy of a desogestrel-only pill compared with a traditional POP, POPs are still not widely used due to an unpredictable bleeding pattern. A new POP containing 4 mg drospirenone has been developed with a 24/4 intake regimen which may improve the bleeding pattern. The objectives of this study were to investigate ovulation inhibition with the new drospirenone-only pill in comparison with the desogestrel-only pill and, in addition, to assess the effects on cervical mucus permeability and bleeding. METHODS: Sixty-four healthy volunteers with proven ovulatory cycles were randomised and treated with either the drospirenone-only or the desogestrel-only pill during two 28-day cycles. Follicular diameter, endometrial thickness, and serum estradiol (E2) and progesterone concentrations were measured and Hoogland scores were determined. Additionally, cervical mucus scores, bleeding and return of ovulation were assessed. RESULTS: Both treatments effectively inhibited ovulation. Follicular diameter, E2 levels and Hoogland scores were equal, demonstrating efficient ovarian suppression. One subject in each group had a Hoogland score of 6, but the criteria for normal luteal activity were not fulfilled. In both groups, ovulation did not occur before day 9 of the post-treatment cycle. Cervical mucus permeability was suppressed in both groups. The median number of bleeding and spotting days was lower in the drospirenone group. CONCLUSIONS: The new drospirenone-only pill inhibited ovulation as effectively as the desogestrel-only pill despite the 4-day hormone-free interval.

Induction and inhibition of crystallization in drug-in-adhesive-type transdermal patches.

PURPOSE: To screen crystallization inhibitors, perform accelerated stability testing and predict saturation solubility of levonorgestrel in drug-in-adhesive patches. METHODS: Differential scanning calorimetry (DSC) studies were compared against slide crystallization studies for screening additives. Release studies were performed from crystallized and supersaturated patches. Die cutting was used for accelerated stability testing of patches. Time lag experiments were performed to predict saturation solubility of levonorgestrel in acrylate adhesive, DuroTak-2516. RESULTS: DSC studies indicated poloxamer to be the best additive whereas slide crystallization studies showed polyvinylpyrrolidone to be better. Supersaturated patches showed higher release profiles relative to crystallized patches. Crystals were observed in crystallized patches even after 96 h of release studies. Die-cutting of patches helped in development of crystals in less time as compared to uncut sheets indicating its usefulness in accelerated stability testing. Saturation solubility of levonorgestrel in DuroTak-2516 was predicted to be 0.09% w/w which was in close agreement with value of 0.1% w/w from solubility calculator on vendor's website. CONCLUSIONS: Crystallization was shown to have negative impact on drug release and patch performance. Slide crystallization studies, die cutting and time lag experiments can be used as tools to help stabilize the otherwise unstable patches.

Ethinylestradiol and levonorgestrel preparations on the Belgian market: a comparative study.

Preparations formulated as coated or film-coated tablets, containing levonorgestrel and the combination ethinylestradiol/levonorgestrel, were evaluated in a comparative study. This study comprised in vitro dissolution, assay and content uniformity. The analytical methods were previously validated according to international guidelines. All examined products complied with the postulated requirements.

Metabolism of the synthetic progestogen norethynodrel by human ketosteroid reductases of the aldo-keto reductase superfamily.

Human ketosteroid reductases of the aldo-keto reductase (AKR) superfamily, i.e. AKR1C1-4, are implicated in the biotransformation of synthetic steroid hormones. Norethynodrel (NOR, 17α-ethynyl-17ß-hydroxy-estra-5(10)-en-3-one), the progestin component of the first marketed oral contraceptive, is known to undergo rapid and extensive metabolism to 3α- and 3ß-hydroxymetabolites. The ability of the four human AKR1C enzymes to catalyze the metabolism of NOR has now been characterized. AKR1C1 and AKR1C2 almost exclusively converted NOR to 3ß-hydroxy NOR, while AKR1C3 gave 3ß-hydroxy NOR as the main product and AKR1C4 predominantly formed 3α-hydroxy NOR. Individual AKR1C enzymes also displayed distinct kinetic properties in the reaction of NOR. In contrast, norethindrone (NET), the Δ(4)-isomer of NOR and the most commonly used synthetic progestogen, was not a substrate for the AKR1C enzymes. NOR is also structurally identical to the hormone replacement therapeutic tibolone (TIB), except TIB has a methyl group at the 7α-position. Product profiles and kinetic parameters for the reduction of NOR catalyzed by each individual AKR1C isoform were identical to those for the reduction of TIB catalyzed by the respective isoform. These data suggest that the presence of the 7α-methyl group has a minimal effect on the stereochemical outcome of the reaction and kinetic behavior of each enzyme. Results indicate a role of AKR1C in the hepatic and peripheral metabolism of NOR to 3α- and 3ß-hydroxy NOR and provide insights into the differential pharmacological properties of NOR, NET and TIB.

Occurrence, fate and removal of synthetic oral contraceptives (SOCs) in the natural environment: a review.

Synthetic oral contraceptives (SOCs) are a group of compounds with progestagenic and/or androgenic activities, with some also possessing estrogenic activities. Recent research has documented that some of these emerging contaminants have adverse effects on aquatic organisms at very low concentrations. To facilitate the evaluation of their latent risks, published works on their occurrence and fate in the environment are reviewed. Androgenic/progestagenic relative potencies or relative binding affinity of these SOCs as well as their physicochemical properties and toxicity are summarized. Appropriate analytical methods are outlined for various environmental sample types, including methods of sample preparation and limit of detection/quantification (LOD/LOQ). Finally results on their occurrence and fate in wastewater treatment plants (WWTPs) and other environments are critically examined.

A study of some practical aspects of high temperature liquid chromatography in pharmaceutical applications.

In the pharmaceutical industry fast and efficient separation techniques play an increasing role among analytical methods because the samples to be investigated grow both in complexity and number, and there is an increasing time pressure to complete the analysis. Reducing the analysis time without decreasing the efficiency is possible using higher pressures, elevated temperatures, smaller particle sizes, or a combination of these approaches. Recently developed chromatographic techniques such as the UHPLC (ultra high performance liquid chromatography) and HTLC (high temperature liquid chromatography) are highly promising in meeting these demands. In this study, high temperature liquid chromatography (HTLC) with a zirconia-based column and temperatures elevated up to 150°C was used. We investigated the chromatographic behaviour of a steroid active pharmaceutical ingredient (levonorgestrel) and its structurally related impurities as model compounds. The effect of the temperature in the range of 50-150°C and the flow-rate in the range of 0.5-3.0 ml/min, and using methanol as an organic modifier, were studied for optimisation of the separation method.

Preparation and characterization of an immunoaffinity chromatography column for the selective extraction of trace contraceptive drug levonorgestrel from water samples.

The preparation and characterization of an immunoaffinity chromatography (IAC) column for the specific extraction and enrichment of trace contraceptive drug levonorgestrel (LNG) from water samples were described. The IAC column was constructed by covalently coupling specific polyclonal antibody against LNG to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. The extraction conditions including loading, washing and eluting solutions, as well as the effect of flow rate on the extraction were carefully optimized. Pure water, 5% of methanol and 50% of methanol were respectively selected as loading, washing and eluting solutions, while the flow rates in the loading, washing and eluting steps were selected to be 1.0, 2.0 and 0.5 mL min(-1), respectively. Under optimal conditions, the IAC column was characterized in terms of maximum capacity, extraction recovery and stability. It was found that, for IAC column packed with 0.2g of solid support immobilized with antibody, the maximum capacity for LNG was about 260 ng. The extraction recoveries of the column for LNG at three different spiked concentrations were within 95.3-106.9%. After more than 35 times repeated usage, there was not significant loss of specific recognition. Using high performance liquid chromatography (HPLC) as an analytical tool, trace amount of LNG in the range of ng L(-1) was found in river water and wastewater samples after 600-fold enrichment, demonstrated the feasibility of the prepared IAC column for LNG extraction.

Voltammetry and quantification of the contraceptive drug norethisterone in bulk form and pharmaceutical formulation.

The electrochemical behavior of norethisterone at the mercury electrode was studied in the universal buffer of various pH values using dc-polarography, cyclic voltammetry and controlled-potential electrolysis. Norethisterone was reduced at the mercury electrode via the consumption of two electrons corresponding to reduction of the 3-keto-delta-4-group in the A-ring of the molecule. The pK(a) value (8.7) of norethisterone was determined from the polarographic and spectrophotometric measurements. A fully validated, simple, sensitive, precise and inexpensive square-wave adsorptive cathodic stripping (SWAdCS) voltammetry procedure was described for trace quantification of bulk norethisterone. The stripping voltammetry peak current of norethisterone in a universal buffer of pH 5 following its accumulation onto the hanging mercury drop electrode (HMDE) at -0.6 V (versus Ag/AgCl/KCl(s)) for 130 s showed a linear response with the concentration over the range 5 x 10(-9) to 3 x 10(-7)M norethisterone. Detection and quantitation limits of 1.5 x 10(-9) and 5 x 10(-9)M bulk norethisterone, respectively, were achieved. The proposed procedure was successfully applied for the assay of norethisterone in Steronate tablets without interference from excipients.

Improved bioavailability of orally administered mifepristone from PLGA nanoparticles.

The objective of this study was to prepare an oral dosage formulation of mifepristone that will improve the oral bioavailability of mifepristone and sustain the release of mifepristone for at least 3 days to effectively control reproduction, especially in coyotes. Nanoparticles containing mifepristone were prepared from dl-lactide/glycolide copolymers (PLGA). Encapsulation efficiency of the nanoparticles was determined by HPLC. In vitro release study was done in 30% isopropyl alcohol in water. In vivo bioavailability study was performed in male rats. Mifepristone and drug-loaded 50/50 PLGA, M(W) 4.4kDa, nanoparticles (equivalent to 100mg/kg mifepristone) were administered orally to rats. The concentration of mifepristone in serum at different time intervals was determined by HPLC. The average sizes of 50/50 PLGA (M(W) 4.4 and 13kDa) nanoparticles containing mifepristone were 516 and 468nm, respectively. The drug encapsulation efficiency was 75.6% at 20% drug loading in 50/50 PLGA (M(W) 4.4kDa) nanoparticles. In vitro cumulative release of mifepristone from the 50/50 PLGA (M(W) 4.4 and 13kDa) nanoparticles with 20% drug loading was 60% and 48% in 72h, respectively. In vivo studies in rats demonstrated that PLGA-1A-nanoparticles increase the bioavailability of mifepristone. We are currently using the nanoparticles containing mifepristone for efficacy studies in coyotes.

Metabolic and clinical effects of progestogens.

Synthetic progestogens differ not only in their hormonal potency, but also in their spectrum of hormonal activities. Beside their progestogenic and anti-oestrogenic effects, they may exert oestrogenic, androgenic, antiandrogenic, glucocorticoid and/or anti-mineralocorticoid activities. Consequently, progestogens may influence various metabolic parameters and modulate oestrogen-induced alterations in lipid metabolism, haemostasis, and various other factors. Progestogens with androgenic properties may counteract ethinyloestradiol (EE)-induced changes in lipoprotein metabolism, but do not cause atherosclerosis in the presence of EE. Oral contraceptives (OCs) containing androgenic progestogens which attenuate the EE-dependent changes in haemostasis, may be associated with a lower risk of venous thromboembolic disease than OCs whose progestogens have a less androgenic profile. Progestogens with androgenic activity may also antagonize oestrogen-induced alterations in various other hepatic proteins and modulate the effect of EE on growth factors. Progestogens with antiandrogenic activity may enhance the beneficial effect of EE in women with hyperandrogenic manifestations. Progestogens with glucocorticoid effects may increase procoagulatory activity in the vessel wall, while progestogens with anti-mineralocorticoid activity may reduce the aldosterone-induced water-retention in some women. For most women the differences in the hormonal pattern of progestogens used in OCs are without clinical relevance, but may be useful for women predisposed for the development of certain disorders.

Biphasic versus monophasic oral contraceptives for contraception.

BACKGROUND: Side effects caused by oral contraceptives discourage compliance with, and continuation of, oral contraceptives. Three approaches have been used to decrease these adverse effects: reduction of steroid dose, development of new steroids, and new formulas and schedules of administration. The third strategy led to the biphasic oral contraceptive pill. OBJECTIVES: To compare biphasic with monophasic oral contraceptives in terms of efficacy, cycle control, and discontinuation due to side effects. Our a priori hypotheses were: (a) biphasic oral contraceptives are less effective than monophasic oral contraceptives in preventing pregnancy; (b) biphasic oral contraceptives cause more side effects, give poorer cycle control, and have lower continuation rates. SEARCH STRATEGY: We searched the computerized databases MEDLINE, EMBASE, POPLINE, LILACS and CENTRAL. In addition, we searched the reference lists of all potentially relevant articles and book chapters. We also contacted the authors of relevant studies and pharmaceutical companies in Europe and the USA. SELECTION CRITERIA: We included randomized controlled trials comparing any biphasic with any monophasic oral contraceptive when used to prevent pregnancy. DATA COLLECTION AND ANALYSIS: We examined the studies found during the various literature searches for possible inclusion and assessed their methodology using Cochrane guidelines. We contacted the authors of all included studies and possibly randomized studies for supplemental information about methodology and outcome. We entered the data into RevMan, and calculated Peto odds ratios for the incidence of intermenstrual bleeding, absence of withdrawal bleeding, and study discontinuation due to intermenstrual bleeding. MAIN RESULTS: Only one trial of limited quality compared a biphasic and monophasic preparation. Percival-Smith 1990 examined 533 user cycles of a biphasic pill (500 microg norethindrone/35 microg ethinyl estradiol for 10 days, followed by 1000 microg norethindrone/35 microg ethinyl estradiol for 11 days; Ortho 10/11) and 481 user cycles of a monophasic contraceptive pill (1500 microg norethindrone acetate/30 microg ethinyl estradiol daily; Loestrin). The study found no significant differences in intermenstrual bleeding, amenorrhea and study discontinuation due to intermenstrual bleeding between the biphasic and monophasic oral contraceptive pills. AUTHORS' CONCLUSIONS: Conclusions are limited by the identification of only one trial, the methodological shortcomings of that trial, and the absence of data on accidental pregnancies. However, the trial found no important differences in bleeding patterns between the biphasic and monophasic preparations studied. Since no clear rationale exists for biphasic pills and since extensive evidence is available for monophasic pills, the latter are preferred.

In vitro release of levonorgestrel from phase sensitive and thermosensitive smart polymer delivery systems.

The objective of this research is to develop injectable controlled delivery systems for the contraceptive hormone, levonorgestrel (LNG), using phase sensitive and thermosensitive polymers. A combination of poly (lactide) (PLA) and a solvent mixture of benzyl benzoate (BB) and benzyl alcohol (BA) was used in the phase-sensitive polymer delivery systems. The effects of solvent systems and polymer concentrations on the in vitro LNG release were evaluated. In the case of thermosensitive polymer delivery systems, a series of low-molecular-weight poly (lactide-co-glycolide)-poly (ethylene glycol)-poly (lactide-co-glycolide) (PLGA-PEG-PLGA) triblock copolymers with varying ratios of lactide/glycolide (LA/GA, 2.0-3.5) were used. The effects of varying block length of copolymers 1, 2, 3, and 4 on the in vitro LNG release were evaluated. Phosphate buffer saline (pH 7.4) containing 0.5% w/v Tween-80 was used as in vitro release medium. The amount of the released LNG was determined by an high pressure liquid chromatography (HPLC) method. A controlled (zero-order) in vitro release of LNG was observed from both phase-sensitive and thermosensitive-polymer delivery systems. Increasing the concentration of the phase-sensitive polymer from 5% to 30% significantly (p < 0.05) decreased the release rate of LNG from 38.32 microg/day to 31.45 microg/day; and increasing the hydrophilic fraction of the solvents mixture (i.e., BA) significantly (p < 0.05) increased the release rate of LNG. In the case of the thermosensitive polymer delivery system, increasing the hydrophobic PLGA block length of copolymers significantly (p < 0.05) decreased the release rate of LNG (98.65 microg/day to 67.60 microg/day). It is evident from this study that both the phase sensitive and thermosensitive polymers are suitable for developing prolonged-release injectable delivery systems for the contraceptive hormone.

Not all progestins are the same: implications for usage.

Recent clinical evidence showing unexpected side-effects of progestins used in contraception and hormone replacement therapy has highlighted the importance of choice of synthetic progestin. The molecular mechanisms of action of the relatively nonspecific and most widely used synthetic progestins, medroxyprogesterone acetate and norethisterone, are discussed in the context of this recent clinical evidence. Future directions involving a more mechanism-based approach for improved therapeutics, with greater specificity and fewer side-effects, are discussed.

Oxidation of bisphenol A, 17beta-estradiol, and 17alpha-ethynyl estradiol and byproduct estrogenicity.

A human breast cancer cell line (MCF-7) was used to investigate the cumulative estrogenicity profiles elicited during the oxidation of three estrogenic compounds [bisphenol A (BPA), 17beta-estradiol (E2), and 17alpha-ethynyl estradiol (EE2)]. High-performance liquid chromatography (HPLC) with a method detection limit (MDL) of approximately 1 nM was used to measure the initial and final concentrations of test compounds during oxidation. Both chlorination and ozonation removed from 75% to >99% of the test compounds in distilled water. Increasing contact time and chlorination dose improved compound removal. Chlorination byproducts of BPA, E2, and EE2 elicited low levels of estrogenicity over an extended period of time. For equivalent molar oxidant dosages, ozone and chlorine had comparable residual proliferative effect values and >99% loss of the parent compounds. For oxidation studies of estrogenic chemicals, ammonium chloride was found to adequately quench residual chlorine without interfering with cell culture assay. Oxidation of test compounds with chlorine and ozone resulted in a similar estrogenicity trend, with a relative higher level of estrogenicity elicited during the early phases of oxidation, which gradually dissipated over the extended exposure time to a stable point. Oxidation with ozone resulted in the rapid transformation of test compounds, reaching a stabilized estrogenic level in 10 min, whereas for chlorination it took more than 120 min for elicited estrogenicity to stabilize.