Comparative Analysis of Microbicidal and Anti-inflammatory Properties of Novel Taurine Bromamine Derivatives and Bromamine T.

Taurine, the most abundant free amino acid in leukocyte cytosol traps hypohalous acids (HOCl and HOBr) to produce N-chlorotaurine (taurine chloramine, NCT and N-bromotaurine (taurine bromamine, Tau-NHBr,) respectively. Both haloamines show anti-inflammatory and antimicrobial properties. However, the therapeutic applicability of Tau-NHBr is limited due to its relatively poor stability. To overcome this disadvantage, we have synthesized the stable N-bromotaurine compounds N-monobromo-2,2-dimethyltaurine (Br-612) and N-dibromo-2,2-dimethyltaurine (Br-422). The aim of this study was to compare anti-inflammatory and microbicidal properties of Br-612 and Br-422 with that of Tau-NHBr and bromamine T (BAT). We have shown that all the tested compounds show similar anti-inflammatory properties. Importantly, the stable N-bromotaurine compounds exerted even stronger microbicidal activity than Tau-NHBr. Finally, for the purpose of topical application of these compounds we have developed a carbomer-based bioadhesive solid dosage form of BAT and Br-612, featuring sustained release of the active substance.

Comparison of the Microbicidal activity of monochloramine and iodine.

UNLABELLED: Recently, we showed that monochloramine (NH2 Cl) has a significantly stronger bactericidal and fungicidal activity than chloramine T despite its lower oxidizing power. This phenomenon was explained by increased penetration because of the higher lipophilicity and smaller bulk of NH2 Cl. As iodine (I2 ) has an even fivefold higher bulk than NH2 Cl, a comparison of both compounds regarding their microbicidal activity became the aim of this study. Aqueous solutions of I2 at a concentration of 10·7 µmol l(-1) killed 10(6) colony forming units per millilitre (CFU ml(-1) ) of Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa to the detection limit of 10(2) CFU ml(-1) within 1 min at 20°C and pH 7·1, while a concentration of 36-355 µmol l(-1) of NH2 Cl was needed to achieve the same effect. Aspergillus fumigatus was inactivated within 5 min by 36 µmol l(-1) I2 and by 355 µmol l(-1) NH2 Cl, Candida albicans within 1 min by 10·7 µmol l(-1) I2 and by 355 µmol l(-1) NH2 Cl. The lipophilicity of I2 , determined with the octanol/water method, was three powers of 10 higher than that of NH2 Cl. The at least 10-fold stronger microbicidal activity of iodine suggests that the hindrance of penetration of the bulky molecule is outweighed by enhanced lipophilicity. SIGNIFICANCE AND IMPACT OF THE STUDY: The microbicidal activity of active halogen compounds increases not only with their reactivity, but also with higher lipophilicity and lower bulk, as shown recently. In this study, iodine showed a higher microbicidal activity than monochloramine and a 1000-fold higher lipophilicity. Therefore, the lipophilicity of a disinfectant may be more important than the bulk for bactericidal activity. These facts should be considered upon the design of new antiseptics and their clinical application.

Superior bactericidal activity of N-bromine compounds compared to their N-chlorine analogues can be reversed under protein load.

AIMS: To investigate and compare the bactericidal activity (BA) of active bromine and chlorine compounds in the absence and presence of protein load. METHODS AND RESULTS: Quantitative killing tests against Escherichia coli and Staphylococcus aureus were performed both in the absence and in the presence of peptone with pairs of isosteric active chlorine and bromine compounds: hypochlorous and hypobromous acid (HOCl and HOBr), dichloro- and dibromoisocyanuric acid, chlorantine and bromantine (1,3-dibromo- and 1,3 dichloro-5,5-dimethylhydantoine), chloramine T and bromamine T (N-chloro- and N-bromo-4-methylbenzenesulphonamide sodium), and N-chloro- and N-bromotaurine sodium. To classify the bactericidal activities on a quantitative basis, an empirical coefficient named specific bactericidal activity (SBA), founded on the parameters of killing curves, was defined: SBA= mean log reductions/(mean exposure times x concentration) [mmol 1(-1) min (-1)]. In the absence of peptone, tests with washed micro-organisms revealed a throughout higher BA of bromine compounds with only slight differences between single substances. This was in contrast to chlorine compounds, whose killing times differed by a factor of more than four decimal powers. As a consequence, also the isosteric pairs showed according differences. In the presence of peptone, however, bromine compounds showed an increased loss of BA, which partly caused a reversal of efficacy within isosteric pairs. CONCLUSIONS: In medical practice, weakly oxidizing active chlorine compounds like chloramines have the highest potential as topical anti-infectives in the presence of proteinaceous material (mucous membranes, open wounds). Active bromine compounds, on the other hand, have their chance at insensitive body regions with low organic matter, for example skin surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The expected protein load is one of the most important parameters for selection of a suited active halogen compound.

Microbicidal activity of monochloramine and chloramine T compared.

Chloramine T (CAT) and monochloramine (NH2Cl) are active chlorine compounds and well-known biocides. CAT has stronger oxidative activity than NH(2)Cl, which is a smaller, more lipophilic molecule. The question arises whether lower oxidative activity can be compensated by higher lipophilicity. To address this problem, we investigated the bactericidal and fungicidal activity of pure NH(2)Cl compared to CAT. Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Aspergillus fumigatus, A. flavus, and Candida albicans were subjected to quantitative killing assays at 20 degrees C and pH 7.1 in equimolar solutions of CAT or NH2Cl. NH2Cl was superior to CAT against all test strains at all test concentrations. At a concentration of 0.036 mM, NH2Cl reduced the count of E. coli (S. aureus) by 3log10 within 1 min (5 min), whereas CAT needed 120 min (30 min) for the same effect. At 0.107 mM NH2Cl, a 3log10 reduction of P. aeruginosa was achieved after 5 min compared to 20 min using CAT. NH2Cl (0.355 mM) caused a 2log10 reduction of C. albicans within 30 s, whereas 60 min were necessary for the same reduction with 0.355 mM CAT. The difference between the antiseptics was even more pronounced when tested on aspergilli. NH2Cl had a significantly stronger bactericidal and fungicidal activity than CAT despite its lower oxidative activity. This phenomenon can be attributed to its lipophilicity and smaller bulk, and it should be taken into account when developing and using chloramine antiseptics.

Effect of N-chlorotaurine mouth rinses on plaque regrowth and plaque vitality.

The purpose of this 4-day plaque regrowth study was to assess the effect of N-chlorotaurine (NCT) mouth rinses on plaque inhibition and plaque vitality. Eighty volunteers participated in this investigator-blind, randomized, clinical controlled study in parallel groups. No oral hygiene was permitted except rinsing with a 2% or 3% NCT mouth rinse, a positive or a negative control. Primary parameters were the plaque index (Silness and Löe, Acta Odontol Scand, 22:121-135, 1964) and plaque vitality (Netuschil et al., J Clin Periodontol, 16:484-488, 1989) after the final rinse. In addition, another plaque index (Turesky et al., J Periodontol, 41:41-43, 1970), plaque area, and bleeding on probing were recorded. All parameters were taken at baseline and day 5. U test was applied on a 5% error level. No differences in plaque inhibition were found between the two NCT formulations and the negative control. However, a statistically significant reduction of plaque vitality compared to the negative and positive control was observed. Discoloration of the tongue and unpleasant taste were recorded in participants in the NCT groups. NCT mouth rinses did not inhibit plaque regrowth, but they did reduce the vitality of plaque bacteria. Methods of prolonging the substantivity of the NCT mouth rinses should be investigated to enhance the antibacterial properties of these formulations.

Tolerability and efficacy of N-chlorotaurine in comparison with chloramine T for the treatment of chronic leg ulcers with a purulent coating: a randomized phase II study.

BACKGROUND: The well-known active chlorine compound chloramine T (CAT) with broad-spectrum antimicrobial activity is in common therapeutic use for leg ulcers with purulent coatings; however, this treatment is painful. The tolerability of the less aggressive N-chlorotaurine (NCT), an endogenous compound also produced in vivo by stimulated human granulocytes, could be superior. OBJECTIVES: To assess the tolerability and efficacy of NCT in the cleaning of purulent coatings in chronic leg ulcers in comparison with CAT. METHODS: In a double-blind, randomized phase IIb clinical study 40 patients were treated for a median of 7 days (range 3-14) with a 1% aqueous solution of either NCT (20 subjects) or CAT (20 subjects) by twice-daily application of dressings soaked in the test solutions. Criteria for evaluation of tolerability were intensity and duration of pain caused by the ulcer therapy and scores of tissue toxicity (necrosis, granulation tissue and re-epithelialization). Therapeutic efficacy was graded as scores of intensity of purulent coating of the ulcers. RESULTS: The concentration tolerated in vitro by human epidermoid carcinoma cells was at least 10-fold higher for NCT (0.01%) compared with CAT (0.0001-0.001%). There was significantly less pain caused by NCT compared with CAT (P < 0.05) on days 1 and 4 and a trend for a shorter duration of pain (P = 0.093). The scores of intensity of coating improved without difference in both treatment groups, whereas granulation and re-epithelialization appeared earlier in the NCT group (P < 0.05). Non-quantitative microbiological cultures from ulcer smears revealed persistence of colonization by bacterial species in approximately half of both treatment groups. CONCLUSIONS: Both active chlorine compounds were helpful in reducing purulent coatings. Because of its lower toxicity and better tolerability, NCT is of advantage in the treatment of leg ulcers.

Enhanced fungicidal activity of N-chlorotaurine in nasal secretion.

The antifungal activity of N-chlorotaurine (NCT), a long-lived oxidant produced by stimulated human leucocytes, was investigated. Incubation of Aspergillus spp., Candida spp., Fusarium spp., Penicillium spp. and Alternaria spp. in 1% NCT (55 mM) for 1-4 h produced a log10 reduction in cfu of between 1 and 4. In samples of nasal secretion, killing was significantly hastened (30 min), which may be explained by the formation of monochloramine by halogenation of ammonium, which was found at a concentration of 1 mM in these samples. For these reasons, NCT is of interest as a new agent for treatment of local inflammatory mycosis, e.g. eosinophilic fungal rhinosinusitis.

N-chlorotaurine, a novel endogenous antimicrobial agent: tolerability testing in a mouse model.

OBJECTIVE: To investigate the tolerability of N-chlorotaurine, a new antimicrobial agent, by application to the middle ear in a mouse model. METHODS: Five BALB/c mice were each injected through the tympanic membrane with 5 microL of 0.1%, 1.0%, and 10% N-chlorotaurine and compared with animals in which 0.9% isotonic sodium chloride solution, 0.2% gentamicin sulfate, and 0.25% trimethyltin chloride were instilled. Auditory brainstem responses to clicks were evaluated repeatedly between 4 and 75 days after injection, and histologic investigations of the inner ear were performed subsequently. Three additional groups of mice were injected with isotonic sodium chloride solution, 1.0% N-chlorotaurine, and 0.25% trimethyltin, and brainstem responses to tone bursts of 8, 16, and 32 kHz were tested. In addition, the middle ear was examined histologically. RESULTS: Mice treated with isotonic sodium chloride solution, 0.1% N-chlorotaurine, and 0.2% gentamicin sulfate did not show changes in response threshold. Treatment with 1.0% and 10% N-chlorotaurine caused a reversible increase in auditory brainstem response threshold by 20 dB 4 days after application because of local irritation around the perforation of the tympanic membrane. In contrast, 0.25% trimethyltin showed a permanent elevation of auditory brainstem response threshold of 10 to 15 dB and a scattered loss of outer hair cells predominantly in the apical turn. No alterations of the inner ear were observed in the other treatment groups. The mucous membrane of the middle ear remained unaffected in all test groups. CONCLUSION: Application of N-chlorotaurine to the middle ear is well tolerated without adverse effects and may be a useful new endogenous antimicrobial agent for local treatment of otologic infections.

The influence of plasma on the disinfecting activity of the new antimicrobial agent N-chlorotaurine-sodium in comparison with chloramine T.

The phenomenon of increasing bactericidal activity of N-chlorotaurine in the presence of chlorine-consuming material has been investigated both on a chemical-analytical and microbiological basis using plasma as substrate and chloramine T for comparison. Chlorine consumption assessed by iodometric titration showed a biphasic time-course with a very fast loss of oxidation capacity within one minute (N-chlorotaurine: -9.3%, chloramine T: -16.8%) followed by a slow loss which could still be detected after 24 h (total loss -61.7% and -74.1%, respectively). Killing curves revealed that an increase in bactericidal activity, in spite of improved consumption, did not occur with all strains, and could be detected only at a certain degree of consumption. Escherichia coli and Pseudomonas aeruginosa showed the most pronounced effect, Streptococcus pyogenes and Proteus mirabilis a medium-sized one, while it was absent in Staphylococcus aureus. With chloramine T, an increase in bactericidal activity could not be proved. The chemical basis of these consumption effects can be reduced to four reaction types: oxidation of thiols; chlorine substitution of activated C-H compounds; transhalogenation; and hydrolytic degradation of N-chloro-alpha-amino acids and -peptides emerging by transhalogenation. The initial fast loss of oxidation capacity can be attributed mainly to oxidation of thiols, while the subsequent slower decrease is caused by the other types of reaction. The increase in bactericidal activity, on the other hand, can be explained by transhalogenation, leading to the formation of more bactericidal N-chloro compounds by which the loss of N-chlorotaurine is over-compensated.

Bactericidal activity of micromolar N-chlorotaurine: evidence for its antimicrobial function in the human defense system.

N-Chlorotaurine, the main representative of long-lived oxidants found in the supernatant of stimulated granulocytes, has been investigated systematically with regard to its antibacterial activity at different physiological concentrations for the first time. N-Chlorotaurine (12.5 to 50 microM) demonstrated a bactericidal effect i.e., a 2 to 4 log(10) reduction in viable counts, after incubation at 37 degrees C for 6 to 9 h at pH 7.0, which effect was significantly enhanced in an acidic milieu (at pH 5. 0), with a 3 to 4 log(10) reduction after 2 to 3 h. Moreover, bacteria were attenuated after being incubated in N-chlorotaurine for a sublethal time, as demonstrated with the mouse peritonitis model. The supernatant of stimulated granulocytes exhibited similar activity. Transmission electron microscopy revealed changes in the bacterial cell membrane and cytoplasmic disintegration with both reacting systems, even in the case of a mere attenuation. The results of this study suggest a significant bactericidal function of N-chlorotaurine and other chloramines during inflammation.

Tolerance of N-chlorotaurine, a new antimicrobial agent, in infectious conjunctivitis - a phase II pilot study.

N-Chlorotaurine (NCT) is an endogenous microbicidal oxidant. This open pilot study (phase IIa) with 9 patients was done to gain first knowledge on the tolerance of NCT in infectious conjunctivitis. By application of 1% NCT 5 times a day, no adverse effects could be observed. All 6 subjects with bacterial conjunctivitis were cured within 3-5 days. Two subjects with epidemic keratoconjunctivitis were treated for 7-10 days and 1 subject with herpes simplex blepharitis for 3 days with no rapid improvement but probable mitigation of inflammation. Therefore, NCT seems to be useful in the treatment of infectious conjunctivitis, and further investigation on its therapeutic efficacy is suggested.

Properties of an enzyme-based low-level iodine disinfectant.

An enzyme-based iodine (EBI) disinfectant that continuously generates free molecular iodine in a controlled fashion was developed and evaluated for use in disinfecting flexible fibreoptic endoscopes (FFEs). EBI is a powder concentrate that produces iodine from sodium iodide and calcium peroxide when catalyzed by horseradish peroxidase. After dissolution in water, it delivers relatively high concentrations of free molecular iodine (> 15 ppm) at relatively low concentrations of total iodine (30-40 ppm). It demonstrates the ability to function as an effective low level iodine disinfectant by rapidly inactivating bacteria, fungi and viruses. A unique feature of the EBI system is the ability to reoxidize reduced iodine which results in a constant level of active (free molecular) iodine during use. EBI inactivates Mycobacterium bovis var BCG more rapidly than 2% glutaraldehyde (Cidex-7). Its sporicidal activity, however, was found to be slower than the aldehyde formulation. The qualification of EBI for use as a practical disinfectant was shown by its negligible toxicity in dermal, ocular, oral and inhalation studies on animals, which is attributed to the low level of total iodine in the solution.

Iodine and disinfection: theoretical study on mode of action, efficiency, stability, and analytical aspects in the aqueous system.

Although they have been in use for nearly 170 years, the mode of action of iodine-based disinfectants is not yet clearly understood, as is manifested, for example, in diverging judgements about the relevance of the individual iodine species. Although studies based on calculated equilibrium concentrations in pure iodine solutions have already been done, there is a lack of knowledge about iodine solutions in the presence of additional iodide which would be of intrinsic importance for disinfection practice. Therefore, a re-calculation was undertaken considering variations of this parameter in the pH range 0-14. The presented calculations concern fresh iodine solutions not affected by disproportionation (iodate formation) and provide information about the equilibrium concentrations of the species I, I2, I3, I5-, I6(2-), HOI, O1-, HI2O-, IO2- and H2OI+. Additional iodide and the pH value have a very pronounced influence on the individual equilibrium concentrations (several powers of ten); hence, conditions can be indicated where the number of species of virtual importance is drastically reduced. In the most common case with iodine in the presence of additional iodide at pH < 6, only I-, I2 and I3- play a role. In the absence of additional iodide, at pH 8-9 and at high dilution (c(I2) < 10(-5) M), on the other hand, HOI accounts for over 90% of the oxidation capacity. At high iodide concentration (e.g., Lugol's solution) the species I5- and I6(2-) make up 8.2% of the oxidation capacity. The iodine cation H2OI+, frequently quoted as an active agent in disinfection, is without any relevance under the conditions occurring in practice, as are IO- and HI2O- which become important only at pH > 10. The stability problem (i.e. rate of iodate formation) arising at pH > 6 can be reduced to hypoiodous acid, as manifested in the simple rate law d[IO3]/dt = 0.25 [HOI]3/[H+] which allows an estimation of stability under weakly alkaline conditions. The results of this study allow us to deduce general qualities of aqueous iodine solutions, such as reactivity, stability, and analytical aspects, and to estimate major disinfection-orientated properties such as microbicidal activity, irritation, and incorporation effects. Though the calculations consider primarily preparations devoid of polymeric organic compounds capable of complexing iodine species, the results can be largely transferred to iodophoric preparations.

The postantibiotic effect of N-chlorotaurine on Staphylococcus aureus. Application in the mouse peritonitis model.

This study was designed to investigate the delay of regrowth (postantibiotic effect) in the presence of N-chlorotaurine (NCT), an endogenous active N-chlorine compound, of Staphylococcus aureus, strain Smith diffuse. The low reactivity of NCT enabled clear temporal separation of the postantibiotic and killing effect to be defined. Delay of regrowth proved to be dependent both on concentration of NCT, and incubation time. The maximum delay was 3 h. Using the model of lethal staphylococcal peritonitis in mice, in-vivo delay of regrowth of bacteria pretreated with N-chlorotaurine could be demonstrated to correlate with survival. It is concluded that the postantibiotic effect of N-chlorotaurine could be an important factor on decreasing virulence of bacteria. This effect was observed after relatively short incubation times.

Tolerance and bactericidal action of N-chlorotaurine in a urinary tract infection by an omniresistant Pseudomonas aeruginosa.

N-chlorotaurine, a weak antimicrobial oxidant produced by stimulated human leukocytes, was used to treat cystitis caused by an omniresistant Pseudomonas aeruginosa. A 21-year-old male patient was treated by repeated daily lavages of the urinary bladder with an aqueous solution of 1% N-chlorotaurine for one month. N-chlorotaurine was well tolerated; no local or systemic side effects could be detected. Despite killing of > 10(6) cfu/ml of bacteria within ten minutes in vitro and in vivo, it was not possible to eradicate the Pseudomonas infection obviously caused by inflammation of the upper urinary tract and perpetuated by intravesical concrements. Nevertheless, in actually localized infection, treatment with N-chlorotaurine might be successful because of its sufficient bactericidal action.

Tolerance of N-chlorotaurine, an endogenous antimicrobial agent, in the rabbit and human eye--a phase I clinical study.

N-chlorotaurine (NCT), an essential weak oxidative N-chloro compound produced by stimulated human leukocytes, shows bactericidal, fungicidal, virucidal and vermicidal efficacy. A double-blind, randomized and placebo controlled study was done to evaluate the tolerance of the aqueous NCT solution by application to rabbit and human conjunctiva. In six rabbits treated with 1% and 3% NCT regimen for nine days no ocular and behaviour changes could be observed. In a pilot study with two volunteers, treatment with 2.8% NCT for five days caused a self-limited conjunctival injection of one subject, while 1% NCT was well tolerated. Subsequently, eight healthy volunteers participated in a phase I clinical study. One percent NCT was applied for five days and was well tolerated by all subjects except for minimal eye burning after the application. Because of these positive results, usage of the antimicrobial agent NCT in ophthalmology is suggested.

Activity of N-chlorotaurine against herpes simplex- and adenoviruses.

N-chlorotaurine, an essential weak oxidant produced by stimulated human leukocytes, is known to have bactericidal, fungicidal and vermicidal properties. This study for the first time demonstrates its virucidal activity. By viral suspension tests at incubation times between 5 and 60 min, virus titers of both Herpes simplex virus type 1 and 2 were reduced about 1.3-2.9 log10 and 2.8-4.2 log10 by 0.1 and 1%, (5.5 and 55 mM) N-chlorotaurine, respectively. Virus titer reduction of adenovirus type 5 between 15 and 60 min was 0.5-2.0 and 0.6-4.0 log10, respectively, by the same concentrations of N-chlorotaurine. These findings support a contribution of N-chlorotaurine in destruction of pathogens during inflammatory reactions and also the possibility of its application as an antiviral agent in human medicine.

Which conditions promote a remanent (persistent) bactericidal activity of chlorine covers?

The interaction of disinfectants containing active chlorine compounds (hypochlorite, chloramine T) with skin surfaces produces a so called chlorine cover which is a true chemical transformation of the protein matrix of the outermost horny skin layer by forming covalent N-Cl bonds. Though its intensity can easily be measured, the results on its remanent (persistent) bactericidal action until now have not been described consistently. While Gottardi and Karl observed a well-defined remanent action which correlated with the cover strength in the artificially with E. coli contaminated arm, Koller, Rotter, and Gottardi were not able to prove it, neither in artificially contaminated hands nor in the case of the resident bacterial colonization. To clarify these not consistent results, the bactericidal activity of chlorine covers on transient (dry contamination of the finger tips with Micrococcus luteus) as well as on resident colonization (after wearing gloves for 3 h) has been re-investigated using contact plates for recovering the bacteria. The results are showing that the remanent bactericidal action of chlorine covers strongly depends on the skin milieu, with moist conditions caused by sweat (surgical gloves) favoring the bactericidal activity. Elution experiments prove that protein constituents (detected by UV-spectrometry) continuously are transported to the skin-surface. It is obvious that the latter equilibrate with the chlorine cover by forming bactericidal N-Cl compounds with the effect that oxidation capacity fixed at the protein matrix is mobilized enabling the remanent bactericidal action.

Rapid killing of Mycobacterium terrae by N-chlorotaurine in the presence of ammonium is caused by the reaction product monochloramine.

We have studied the activity of the weak endogenous oxidant N-chlorotaurine against Mycobacterium terrae. The study revealed slow killing of more than 2h duration by 1% (55 mM) N-chlorotaurine. In the presence of ammonium chloride, however, killing times decreased to a few minutes, even by 0.1% N-chlorotaurine. This phenomenon is explained by formation of the lipophilic and therefore more bactericidal monochloramine as a result of transhalogenation of ammonia by N-chlorotaurine.

[Can the DGHM test for the surgical hand disinfection be used to detect residual effects?]. / Eignet sich die DGHM-Prüfmethode für die Chirurgische Händedesinfektion zum Nachweis einer remanenten Wirkung?

It is investigated if the log reductions measured by the DGHM test for examination and evaluation of disinfecting procedures for the surgical hand wash, i.e. the short and long term values RFreference, 0h, RFreference, 3h, RFpreparation, 0h and RFpreparation, 3h can be used to detect residual effects (a remanent action) of the tested preparation. To do this the differences delta RF = RF0h-RF3h of reference and tested preparation have been formed which are a measure for the bacterial regeneration rate. A remanence index RI = magnitude of delta RFreference/magnitude of delta RFpreparation has been specified, which, in case RI > 1 indicates a residual effect of the test preparation in relation to the reference. The evaluation of 21 testing protocols is showing that the calculated remanence indexes are far away from any significance. This is attributed on the one hand to the great standard deviations of the log reductions and on the other hand to virtually only small residual effects of the tested preparations.