The EU Biocidal Products Directive and the consequences for Dinsinfectants

The title of my presentation suggests or at least anticipates that the EU legislative process, to provide a harmonised regulatory platform for biocides in the EU, has been finalised when this symposium takes place.When adapted and subsequently implemented in the EU member states (estimation Q3 1999), this directive will have a major impact on the market of disinfectants for both the suppliers of active substances and formulators supplying disinfectant products to customers and consumers. This presentation will focus on (technical) regulatory consequences rather than economical or business effects.

• • scope: which products will fall under the regime of this directive, which are excluded?

• • principle: one product—one authorization and mutual recognition; lead times involved?

• • procedure: how will the simplified procedures foreseen (hopefully) help the disinfectant supplier?

• • data requirements: why do the dossier requirements for products and particularly actives need to be so excessively costly and what is the role of risk assessment?

• • transitional arrangements: what can and can't disinfectant suppliers do to keep their products on the market or to introduce new products?

• • review programme: what will happen to the availability of active substances?

The directive itself will not give sufficiient answers to all issues mentioned above to allow a harmonised interpretation across the EU. A number of technical guidance documents is therefore being developed covering Common Principles, Data Requirements and Conditions to include an Active Substance on Annex I. The workability of the whole directive will very much be dependent on the practical details to be dealt with in these documents.Disinfectant suppliers also do recognize the merits of the directive (improved protection of man and the environment, harmonisation across EU, etc.), provided however that it does not undermine the efforts of our industry to deliver better hygiene with safe, effective but also affordable disinfectants.     

Resistance mechanisms of bacteria to antimicrobial compounds

A range of antimicrobial compounds (bactericides) commonly termed biocides, microbicides, sanitizers, antiseptics and disinfectants are available, all of which are claimed by their producers to kill bacteria. Resistance has been defined as the temporary or permanent ability of an organism and its progeny to remain viable and/or multiply under conditions that would destroy or inhibit other members of the strain. Bacteria may be defined as resistant when they are not susceptible to a concentration of antibacterial agent used in practice. Traditionally, resistance refers to instances where the basis of increased tolerance is a genetic change, and where the biochemical basis is known. Antimicrobial substances target a range of cellular loci, from the cytoplasmic membrane to respiratory functions, enzymes and the genetic material. However, different bacteria react differently to bactericides, either due to inherent differences such as unique cell envelope composition and non-susceptible proteins, or to the development of resistance, either by adaptation or by genetic exchange. At low concentrations bactericides often act bacteriostatically, and are only bacteriocidal at higher concentrations. For bactericides to be effective, they must attain a sufficiently high concentration at the target site in order to exert their antibacterial action. In order to reach their target site(s), they must traverse the outer membrane of the gram negative bacteria. Bacteria with effective penetration barriers to biocides generally display a higher inherent resistance than those bacteria which are readily penetrated. The rate of penetration is linked to concentration, so that a sufficiently high bactericide concentration will kill bacteria with enhanced penetration barriers. It has been indicated that susceptible bacterial isolates acquire increased tolerance to bactericides following serial transfer in sub-inhibitory concentrations. Whereas the basis of bacterial resistance to antibiotics is well know, that of resistance to antiseptics, disinfectants and food preservatives is less well understood.Three mechanisms of resistance that have been reported include:

• limited diffusion of antimicrobial agents through the biofilm matrix,

• interaction of the antimicrobial agents with the biofilm matrix (cells and polymer),

• enzyme mediated resistance,

• level of metabolic activity within the biofilm

• genetic adaptation

• efflux pumps and

• outer membrane structure.

     

Polarity proteins regulate mammalian cell–cell junctions and cancer pathogenesis

Polarity pathways regulate important functions during the formation and maintenance of cell–cell junctions and during morphogenesis. In addition, cell polarity pathways are emerging as critical regulators of initiation and progression of carcinoma by functioning as tumor suppressors, downstream of oncogenes, or promoters of the metastatic process (Figure 2). It is highly likely that further analysis of cell polarity proteins and the pathways they control will identify novel biomarkers and potential drug targets for managing and treating patients with carcinoma.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest

•• of outstanding interest

The effects of melanocortin agonists and antagonists on leptin-induced fever in rats

1. Over three experiments, separate groups of adult male Sprague–Dawley rats received intracerebroventricular (ICV) injections of either vehicle, recombinant rat leptin (1 μg), or leptin (4 μg), then two ICV injections, 30 min apart of vehicle/vehicle, leptin (4 μg)/vehicle, vehicle/α-MSH (300 ng), or leptin/α-MSH, and then vehicle/vehicle, leptin (4 μg)/vehicle, vehicle/ SHU-9119 (200 ng; a MC 3/4 receptor antagonist), or leptin/SHU-9119. Core temperatures (T_c), food intake and body weights were monitored.

2. Four microgram leptin resulted in the induction of fever, an effect blocked by injection of α-MSH. Antagonism of MC 3/4 receptors with SHU-9119 did not augment leptin-induced fever, but did block the inhibitory actions of leptin on food intake.

3. These data demonstrate the inhibitory effects of exogenous α-MSH on leptin-induced fever, but suggest that endogenous melanocortin action at MC 3/4 receptors does not tonically inhibit febrigenesis caused by leptin administration.

Metabolic rate, maximum metabolism, and advantages of torpor in the fat mouse Steatomys pratensis natalensis Roberts, 1921 (Dendeomurinae)

1. The fat mouse Steatomys pratensis natalensis (mean body mass 37.4±0.43 (se)) has a low euthermic body temperature T_b=30.1–33.8 °C and a low basal metabolic rate (BMR)=0.50 ml O₂ g⁻¹ h⁻¹.

2. Below an ambient temperature (T_a)=15 °C, the mice were hypothermic.

3. The lowest survivable T_a=10 °C.

4. Torpor is efficient in conserving energy between T_a=15–30 °C, below T_a=15 °C, the mice arouse.

5. Euthermic and torpid mice were hyperthermic at T_a=35 °C.

6. Thermal conductance was 0.159 ml O₂ g⁻¹ h⁻¹ °C⁻¹, 98.8% of the expected value.

7. Non-shivering thermogenesis (NST) was 2.196 ml O² g⁻¹ h⁻¹ (3.69×BMR).

8. Maximal oxygen consumption, however, was 3.83 ml O₂ g⁻¹ h⁻¹ (6.44×BMR), indicating that other methods of heat production are additive.

9. Because fat mice conserve energy by torpor only between T_a=15–30 °C, we suggest that torpor may be a more important mechanism for surviving food shortages than for surviving cold weather.

The role of β- and α-adrenoreceptors on blood flow and temperature of brown adipose tissue and involvement of nitric oxide in their effects

1. o

2. 1. Isoproterenol increased interscapular brown adipose tissue (BAT) blood flow and temperature.

3. 2. Phenylephrine increased BAT temperature, but did not affect blood flow. The effect was completely suppressed by N^ω-nitro- -arginine methyl ester ( -NAME), an inhibitor of endogenous NO synthesis.

4. 3. Isoproterenol plus phenylephrine increased BAT blood flow and temperature earlier than isoproterenol alone.

5. 4. CL316,243 increased BAT blood flow and temperature. These effects were also inhibited by -NAME.

6. 5. These data suggest that BAT blood flow as well as thermogenesis is regulated mainly by β-adrenoreceptors and partly by α₁-adrenoreceptors, and NO may be a common mediator for their regulations.

     

Cooled neutrophils become deposited in the microcirculation after infusion: A potential mechanism for microvascular disruption following tissue hypothermia

1. We examined whether changes in rigidity and adhesiveness of neutrophils exposed to cooling and rewarming observed in vitro might impair microvascular perfusion in vivo. Neutrophils from donor rats were fluorescently (calcein-AM) or radioactively (Indium-111) labelled, incubated at 10 or 37 °C in vitro, and infused into recipients. Changes in transit rate and adhesive behaviour within post-capillary venules was quantified in m. extensor digitorum longus (EDL) using intravital microscopy, and tissue distribution determined.

2. There was an increased propensity of cooled cells to undergo adhesion following transfer into the recipient rat. However, cooling had no effect on median transit (354 μm s⁻¹) or rolling (14 μm s⁻¹) velocities during the first 5 min after infusion suggesting that cooling promotes adhesion, but does not delay passage through capillaries. Cooled neutrophils subsequently transformed to stationary adhesion. Their immobilisation was higher than for cells held at 37 °C (P<0.05), and once immobilised they remained firmly adherent to the vessel wall. Cooled, radiolabelled neutrophils showed tissue-specific accumulation after 3 min, but were cleared to the same extent as warmed cells by 20 min.

3. Our data suggest that cooling and rewarming of neutrophils impairs their ability to transit microvessels, reflecting changes in adhesive and mechanical properties observed in vitro, and may contribute to cold-associated circulatory pathology.

Low-temperature tolerance of false codling moth Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) in South Africa

(1) The low-temperature tolerance of false codling moth (FCM) Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a significant aspect of this pest's population dynamics and has implications for post-harvest control and sterile insect technique programs.

(2) Here, we report results of experiments examining the effects of time, temperature and their interactions on low-temperature tolerance in adult FCM. In addition, using a variety of thermal pre-treatments, we examine the potential for hardening responses over several hours to improve low-temperature tolerance in FCM.

(3) Lower lethal temperature assays showed significant effects of time, temperature and significant interactions between time and temperature on survival (p<0.0001 in all cases). The temperature at which the probability of survival of 50% of the FCM population after 2 h of exposure was −4.5 °C, which varied significantly to −0.5 °C in 10 h. Gender and early adult age did not affect low-temperature tolerance of FCM.

(4) Using a range of non-lethal, low- and high-temperature pre-treatments, FCM survival could not be increased (p>0.84) and thus limited evidence for rapid cold hardening was found. These results are discussed with respect to microclimate temperatures in typical FCM environments and have implications for understanding population dynamics in this species and the diversity of low-temperature responses of insects.

Macromolecular conformation of chitosan in dilute solution: A new global hydrodynamic approach

Chitosans of different molar masses were prepared by storing freshly prepared samples for up to 6 months at either 4, 25 or 40 °C. The weight-average molar masses, M_w and intrinsic viscosities, [η] were then measured using size exclusion chromatography coupled to multi-angle laser light scattering (SEC-MALLS) and a “rolling ball” viscometer, respectively.The solution conformation of chitosan was then estimated from:

(a) the Mark–Houwink–Kuhn–Sakurada (MHKS) power law relationship [η] = kM_w^a and

(b) the persistence length, L_p calculated from a new approach based on equivalent radii [Ortega, A., & Garcia de la Torre, J. (2007). Equivalent radii and ratios of radii from solution properties as indicators of macromolecular conformation, shape, and flexibility. Biomacromolecules, 8, 2464–2475].

Both the MHKS power law exponent (a = 0.95 ± 0.01) and the persistence length (L_p = 16 ± 2 nm) are consistent with a semi-flexible rod type (or stiff coil) conformation for all 33 chitosans studied. A semi-flexible rod conformation was further supported by the Wales–van Holde ratio, the translational frictional ratio and sedimentation conformation zoning.     

Benthic foraminiferal associations in the Northern Bay of Safaga, Red Sea, Egypt

In the shallow-water area (0–70 m) of the Northern Bay of Safaga, 73 surface samples were studied with respect to total foraminiferal fauna. The samples cover a great variety of shallow-water environments and yielded 239 foraminiferal taxa. Based on q-mode cluster analysis, and tested by canonical discriminant analysis, the samples are grouped into 13 foraminiferal associations, each characterized by several species:

(1) Quinqueloculina mosharrafai-Borelis schlumbergeri-Brizalina simpsoni Ass.,

(2) Heterostegina depressa-Amphlstegina lessonii/bicirculata Ass.,

(3) Cibicidids-Rosalina-Amphistegina lobifera-Pseudoschlumbergerina ovata Ass.,

(4) Peneroplis planatus Ass.,

(5) Peneroplis planatus-Varidentella neostriatula Ass.,

(6) Peneroplis planatus-Coscinospira hemprichii-Varidentella neostriatula Ass.,

(7) Quinqueloculina spp. Ass.,

(8) Hauerina diversa-Sorites orbiculus Ass.,

(9) Verneuilina sp.-n Articulina pacifica-Reussella simplex Ass.,

(10) Textularia agglutinans/rugulosa-Bolivina variabilis Ass.,

(11) Textularia agglutinans-Challengerella bradyi-Elphidium jenseni/simplex Ass.,

(12) Operculina ammonoides-Adelosina laevigata-Brizalina striatula/subspathulata Ass.,

(13) Bolivina variabilis—Miliolinella-Nonion fabum-Elphidium simplex Ass.

The characteristic forms of each association are linked to specific environmental parameters and modes of life. As long as taxonomic uniformitarianism can be applied, similar associations with comparable ecological demands may also be detected in the fossil record. For comparisons with taxonomically different material the 13 associations are summarized into 4 categories each reflecting a specific type of substrate. In the fossil record these categories may be recognized by general morphological characteristics of the foraminiferal tests and by sedimentological data. The 4 substrate types are: hardground, sand (with or without seagrass and/or corals), firmground, and soft bottom.     

Personal and Household Hygiene,Environmental Contamination,and Health in Undergraduate Residence Halls in New York City, 2011

Background

While several studies have documented the importance of hand washing in the university setting, the added role of environmental hygiene remains poorly understood. The purpose of this study was to characterize the personal and environmental hygiene habits of college students, define the determinants of hygiene in this population, and assess the relationship between reported hygiene behaviors, environmental contamination, and health status.

Methods

501 undergraduate students completed a previously validated survey assessing baseline demographics, hygiene habits, determinants of hygiene, and health status. Sixty survey respondents had microbiological samples taken from eight standardized surfaces in their dormitory environment. Bacterial contamination was assessed using standard quantitative bacterial culture techniques. Additional culturing for coagulase-positive Staphylococcus and coliforms was performed using selective agar.

Results

While the vast majority of study participants (n = 461, 92%) believed that hand washing was important for infection prevention, there was a large amount of variation in reported personal hygiene practices. More women than men reported consistent hand washing before preparing food (p = .002) and after using the toilet (p = .001). Environmental hygiene showed similar variability although 73.3% (n = 367) of subjects reported dormitory cleaning at least once per month. Contamination of certain surfaces was common, with at least one third of all bookshelves, desks, refrigerator handles, toilet handles, and bathroom door handles positive for >10 CFU of bacteria per 4 cm² area. Coagulase-positive Staphylococcus was detected in three participants'' rooms (5%) and coliforms were present in six students'' rooms (10%). Surface contamination with any bacteria did not vary by frequency of cleaning or frequency of illness (p>.05).

Conclusions

Our results suggest that surface contamination, while prevalent, is unrelated to reported hygiene or health in the university setting. Further research into environmental reservoirs of infectious diseases may delineate whether surface decontamination is an effective target of hygiene interventions in this population.     

Acute effect of a single whole-body cryostimulation on prooxidant–antioxidant balance in blood of healthy, young men

1. We have examined the prooxidative–antioxidative reaction to extremely low temperatures (−130 °C) during a one-time cryostimulation in 15 young, clinically healthy individuals.

2. The total lipid peroxides as the total oxidative status (TOS) and the total antioxidative status (TAS) were measured in blood plasma collected in the morning of the day of cryostimulation, 30 min after the cryostimulation, and on the following morning.

3. The level of stress expressed by total oxidative status in plasma, resulting from exposure to extremely low temperatures, was statistically significantly lowered 30 min after leaving the cryochamber than prior to the exposure. The next day, the TOS level still remained lower than the initial values. The TAS level decreased after leaving the cryochamber and remained elevated the following day.

Trade-off between energy budget, thermogenesis and behavior in Swiss mice under stochastic food deprivation

(1) The role of metabolic rate, thermogenesis and behavior in trade-off strategy was examined in Swiss mice under stochastic food deprivation (FD).

(2) For the mice under severe FD, food intake on feeding days increased, and basal metabolic rate (BMR) and nonshivering thermogenesis decreased significantly. The duration of activity decreased but resting behavior increased significantly on feeding days.

(3) It suggested that a trade-off between increased food intake and decreased energy expenditure related to lower BMR, thermogenesis and activity might be employed to meet reduced calorie intake caused by unpredictable FD.

Bioenergetic responses of sub-adult sea cucumber Apostichopus japonicus (Selenka) (Echinodermata: Holothuroidea) to temperature with special discussion regarding its southernmost distribution limit in China

1. This paper investigated the bioenergetic responses of the sea cucumber Apostichopus japonicus (wet weights of 36.5±1.2 g) to different water temperatures (5, 10, 15, 20, 25 and 30 °C) in the laboratory.

2. Results showed that theoretically the optimal temperatures for energy intake and scope for growth (SFG) of sub-adult A. japonicus was at 15.6 and 16.0 °C, respectively. The aestivation threshold temperature for this life-stage sea cucumber could be 29.0 °C by taking feeding cessation as the indication of aestivation.

3. Our data suggests that A. japonicus is thermo-sensitive to higher temperature, which prevents it from colonising sub-tropical coastal zones. Therefore, water temperature plays an important role in its southernmost distribution limit in China.

4. The potential impact of global ocean warming on A. japonicus might be a northward shift in the geographical distribution.

Antioxidative defense organization in retroperitoneal white adipose tissue during acclimation to cold—The involvement of l-arginine/NO pathway

1. Retroperitoneal white adipose tissue (RpWAT) antioxidative defense was investigated in untreated, l-arginine-treated and N^ω-nitro-l-arginine methyl ester (l-NAME)-treated rats kept at 4±1 °C (1, 3, 7, 12, 21 and 45 days) and compared to control rats at 22±1 °C.

2. Cold-acclimation-induced RpWAT weight decrease was accompanied by a decline in glutathione level and increased activity of manganese superoxide dismutase (MnSOD), glutathione S-transferase (GST), catalase, glutathione peroxidase and glutathione reductase at different time-points.

3. l-arginine accelerated RpWAT weight decrease, the increase in MnSOD and GST activities and the prolonged increase of catalase, MnSOD and GST activities. l-NAME delayed cold-induced catalase activity increase and tissue weight decrease. Prolonged l-NAME-treatment had a similar effect on RpWAT as l-arginine.

4. Results suggest the involvement of l-arginine/NO pathway in RpWAT oxidative metabolic augmentation induced by cold-acclimation.

Report from the in vitro micronucleus assay working group

At the Washington “2nd International Workshop on Genotoxicity Testing” (25–26 March 1999) current methodologies and data for the in vitro micronucleus test were reviewed. As a result, guidelines for the conduct of specific aspects of the protocol were developed. Agreement was achieved on the following topics: choice of cells, slide preparation, analysis of micronuclei, toxicity, use of cytochalasin-B, number of doses, and treatment/harvest times [Environ. Mol. Mutagen. 35 (2000) 167]. Because there were a number of important in vitro micronucleus validation studies in progress, it was not possible to design a definitive, internationally harmonized protocol at that time. These studies have now been completed and the data were reviewed at the Plymouth “3rd International Workshop on Genotoxicity Testing” (28–29 June 2002). Data from studies coordinated by the French Society of Genetic Toxicology, Japanese collaborative studies, European pharmaceutical industry validation studies, along with data from Lilly Research Laboratories were used to prepare conclusions on the main aspects of the in vitro micronucleus protocol. In this paper, the consensus agreements on the protocol for performing the in vitro micronucleus assay are presented. The major recommendations concern:

1. Demonstration of cell proliferation: both cell lines and lymphocytes can be used, but demonstration of cell proliferation in both control and treated cells is compulsory for the acceptance of the test.

2. Assessment of toxicity and dose range finding: assessment of toxicity should be performed by determining cell proliferation, e.g. increased cell counts (CC) or population doubling (PD) without cytochalasin-B, or e.g. cytokinesis-block proliferation index with cytochalasin-B; and by determining other markers for cytotoxicity (confluency, apoptosis, necrosis) which can provide valuable additional information.

3. Treatment schedules for cell lines and lymphocytes.

4. Choice of positive controls: without S9-mix both a clastogen (e.g. mitomycin C or bleomycin) and an aneugen (e.g. colchicine) should be included as positive controls and a clastogen that requires S9 for activity when S9-mix is used (e.g. dimethylnitrosamine, or cyclophosphamide in those cell types that cannot activate this agent directly).

5. Duplicate cultures and number of cells to be scored.

6. Repeat experiments: in lymphocytes, for each experiment blood from 2 different healthy young and non-smoking donors should be compared. In cell lines, the experiments need only to be repeated if the first one is negative.

7. Statistics: statistical significance should not be the sole factor for determining positive results. Biological meaning should serve as a guideline. Examples of statistical analyses are given.

     

Pulmonary gas exchange kinetics during exercise: physiological inferences of model order and parameters

1. 1. The ventilatory and pulmonary gas exchange responses during moderate exercise can be appropriately modelled with first-order dynamics.

2. 2. A delay term, reflecting tissue-to-lung transit time, is needed for accurate characterization, however.

3. 3. The O₂ uptake time constant ( reflects the enzymatically controlled tissue O₂ utilization.

4. 4. is appreciably longer than , consequent to the tissue CO₂ capacitance.

5. 5. As typically longer than , transient errors in alveolar and arterial blood gas tensions are predicted: small for P_CO2 but much larger for P_O2.

6. 6. At work rates above the lactate threshold, a slow and delayed component of V̇_O2 induces an additional V̇ component (“excess” V̇_O2), leading to more rapid fatigue.

7. 7. The ventilatory compensation for the metabolic acidemia at these work rates is slow, with compensation being poor for rapid-incremental exercise.

8. 8. A justifiable control model of the coupling of ventilation to metabolism must cohere with these demonstrable physiological characteristics.

Genetic control of arthritis in rats

This study was specifically designed to analyse the genetic control of the chronic disease course for the development of arthritis. Arthritis models with a chronic erosive arthritis are collagen induced arthritis induced with homologous collagen in oil but also arthritis induced with certain non-immunogenic adjuvants such as pristane and avridine. In the presently described experiment we have used pristane induced arthritis. A single injection of 150 μl pristane induces severe chronic arthritis in DA rats. The disease mimics rheumatoid arthritis in many aspects such as the chronic disease course, an erosive inflammation of peripheral joints, symmetric involvement of the joints and the development of rheumatoid factors. To determine the genetic contribution we have used a number of inbred, recombinant inbred and congenic strains as well as specifically designed segregating crosses. An influence by the MHC region (designated Pia1 locus) on the chronic disease course was determined through the uses of MHC congenic LEW strains in which the RT1-f haplotype conferred highest susceptibility. To map genes outside of MHC we used an F2 cross between the highly susceptible DA and the resistant E3 strains. Loci exclusively associated with different phenotypes of the disease could be identified:

Arthritis onset (Pia2 and Pia3).

Severity and joint erosions (Pia4).

Chronicity (Pia5 and Pia6) and Pia1 (determined from MHC congenic (strains)

These findings demonstrates that a chronic self-perpetuative disease, mimicking rheumatoid arthritis, is controlled by different set of genes exclusively linked to different phases of the disease course such as arthritis onset, joint erosions, severity and chronicity.