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TOXSWA calculates exposure concentrations of plant protection products in water and in sediment and isused in risk assessment studies of aquatic organisms. Replacement of the TOXSWA 1.2 software by the morerecent FOCUS_TOXSWA software for use with the NL standard scenarios was investigated. The impact of thereplacement on the calculated exposure concentrations of parent compounds and of metabolites wasdetermined. For parent substances the calculated exposure concentrations of the two TOXSWA versions wereequivalent. Due to a different way to simulate metabolites, the differences in exposure concentrationscalculated by the two versions can be large. For the simulation of metabolite substances two approaches aregiven to handle situations where the formation fraction is not known. TOXSWA berekent blootstellingsconcentraties van gewasbeschermingsmiddelen in water en in sediment voorde risicobeoordeling van aquatische organismen. Onderzocht is of de TOXSWA 1.2 software kan wordenvervangen door de meer recente FOCUS_TOXSWA software om te gebruiken met de NL standaardscenario’s.De invloed van vervanging van TOXSWA 1.2 door FOCUS_TOXSWA 4.4.3 op de berekende blootstellingsconcentratiesvan moederstoffen en van metabolieten is bepaald. De berekende blootstellingsconcentratiesvoor moederstoffen met de twee TOXSWA-modelversies kwamen overeen. Door het verschil in methodiekvan het simuleren van metabolieten kunnen er grote verschillen zijn in de berekende blootstellingsconcentratiesvoor metabolieten berekend door de twee modelversies. Om metabolieten te simuleren,worden twee methoden gegeven voor gevallen waarin de vormingsfractie van de metaboliet niet bekend is.

Background Measurement error in exposure variables is an important issue in epidemiological studies that relate exposures to health outcomes. Such studies, however, usually pay limited attention to the quantitative effects of exposure measurement error on estimated exposure-outcome associations. Therefore, the estimators for exposure-outcome associations are prone to bias. Existing methods to adjust for the bias in the associations require a validation study with multiple replicates of a reference measurement. Validation studies with multiple replicates are quite costly and therefore, in some cases only a single–replicate validation study is conducted besides the main study. For a study that does not include an internal validation study, the challenge in dealing with exposure measurement error is even bigger. The challenge is how to use external data from other similar validation studies to adjust for the bias in the exposure-outcome association. In accelerometry research, various accelerometer models have currently been developed. However, some of these new accelerometer models have not been properly validated in field situations. Despite the widely recognized measurement error in the accelerometer, some accelerometers have been used to validate other instruments, such as physical activity questionnaires, in measuring physical activity. Consequently, if an instrument is validated against the accelerometer, and the accelerometer itself has considerable measurement error, the observed validity in the instrument being validated will misrepresent the true validity. Methodology In this thesis, we adapted regression calibration to adjust for exposure measurement error for a single-replicate validation study with zero-inflated reference measurements and assessed the adequacy of the adapted method in a simulation study. For the case where there is no internal validation study, we showed how to combine external data on validity for self-report instruments with the observed questionnaire data to adjust for the bias in the associations caused by measurement error in correlated exposures. In the last part, we applied a measurement error model to assess the measurement error in physical activity as measured by an accelerometer in free-living individuals in a recently concluded validation study. Results The performance of the proposed two-part model was sensitive to the form of continuous independent variables and was minimally influenced by the correlation between the probability of a non-zero response and the actual non-zero response values. Reducing the number of covariates in the model seemed beneficial, but was not critical in large-sample studies. We showed that if the confounder is strongly linked with the outcome, measurement error in the confounder can be more influential than measurement error in the exposure in causing the bias in the exposure-outcome association, and that the bias can be in any direction. We further showed that when accelerometers are used to monitor the level of physical activity in free-living individuals, the mean level of physical activity would be underestimated, the associations between physical activity and health outcomes would be biased, and there would be loss of statistical power to detect associations. Conclusion The following remarks were made from the work in this thesis. First, when only a single-replicate validation study with zero-inflated reference measurements is available, a correctly specified regression calibration can be used to adjust for the bias in the exposure-outcome associations. The performance of the proposed calibration model is influenced more by the assumption made on the form of the continuous covariates than the form of the response distribution. Second, in the absence of an internal validation study, carefully extracted validation data that is transportable to the main study can be used to adjust for the bias in the associations. The proposed method is also useful in conducting sensitivity analyses on the effect of measurement errors. Lastly, when “reference” instruments are themselves marred by substantial bias, the effect of measurement error in an instrument being validated can be seriously underestimated.

Absorption of toxic substances via the skin is an important phenomenon in the assessment of the risk of exposure to these substances. People are exposed to a variety of substances and products via the skin, either directly or indirectly, while at work, at home or in public space. Pesticides, organic solvents and metalworking fluids are seen to be important contributors to adverse health effects due to occupational exposure via the skin. In daily life, cosmetics, clothing and household products are the most relevant commodities with respect to exposure via the skin. Given the importance of skin exposure in the assessment of the risk of toxic substances, the objective of this thesis was to further develop, evaluate and improve methods for including skin absorption data this assessment. In this thesis, four factors influencing dermal absorption, namely dermal loading (chapters 3 and 6), irritative/corrosive potential (chapters 3 and 4), frequency of exposure (chapters 3, 4 and 5) and the vehicle used (chapter 5), were investigated in more detail. Furthermore, a model to extrapolate infinite dose absorption data to finite dose conditions, baptized Dermal Absorption Model for Extrapolation (DAME), was developed and tested. I n chapter 2 of this thesis, the relationship between relative dermal absorption and dermal loading was investigated. Hundred-and-thirty-eight dermal publicly available absorption experiments with 98 substances were evaluated. The results obtained revealed that dermal loading ranged mostly between 0.001 and 10 mg/cm2. In 87 experiments (63%), an inverse relationship was observed between relative dermal absorption and dermal loading. On average, relative absorption at high dermal loading was 33 times lower than at low dermal loading. Known skin irritating and volatile substances less frequently showed an inverse relationship between dermal loading and relative absorption. It was concluded that when using relative dermal absorption in regulatory risk assessment, its value should be determined at or extrapolated to dermal loadings relevant for the exposure conditions being evaluated. I n chapter 3 of this thesis, a literature search was presented with the aim to investigate whether neglecting the effects of repeated exposure may lead to an incorrect estimate of dermal absorption. The results demonstrated that the effect of repeated versus single exposure does not demonstrate a unique trend. Nevertheless, an increase in daily absorption was frequently observed upon repeated daily exposure. The little information available mostly concerned pharmaceuticals. However, consumers and workers may be repeatedly exposed to other types of chemicals, like disinfectants and cleaning products, which often contain biocidal active substances that may decrease the barrier function of the skin, especially after repeated exposure. These biocidal products, therefore, may present a safety risk that is not covered by the current risk assessment practice since absorption data are usually obtained by single exposure experiments. Consequently, it was decided to investigate the importance of this issue for biocide safety evaluation. As the literature search revealed that hardly any data on absorption upon repeated dermal exposure to biocides are available, it was concluded that data need to be generated by testing. To cover the entire range of biocidal products in such testing, a representative series of biocidal substances should be tested, making in vitro testing of dermal absorption the preferred choice over in vivo testing. Based on an inventory made, it appeared that the 16 product types represented among the biocidal products authorised in the Netherlands could be clustered into 6 more or less homogeneous categories based on similarity in active substances. This result could facilitate experimental testing by providing a basis for selection of a limited number of representative compounds to be evaluated. I n chapter 4 of this thesis, the importance of the effect of repeated dermal exposure on skin permeability for biocide safety evaluation was investigated, using a selection of nine representative biocides from the inventory made in chapter 3. The in vitro dermal penetration of tritiated water and [14C]propoxur was chosen as a measure of the permeability and integrity of human abdominal skin after single and repeated exposure. The results indicated that single and repeated exposure to specific biocidal products (e.g. the quaternary ammonium chlorides DDAC and ADBAC) may significantly increase skin permeability, especially when the compounds are applied at high concentrations, while a substance like formaldehyde may reduce skin permeability under specific conditions. I n chapter 5 of this thesis, the in vitro dermal absorption kinetics of the quaternary ammonium compound didecyldimethylammonium chloride (DDAC) during single and repeated exposure was studied in more detail. In addition, the influence of biocidal formulations on the absorption of DDAC was investigated, because it was expected that formulation characteristics may be another factor influencing its dermal absorption. The analysis of biocidal products on the Dutch market, reported in chapter 3, indicated that DDAC is often used in combination with other active ingredients. DDAC was most frequently combined with formaldehyde, glutaraldehyde and/or alkyldimethylbenzyl­ammo­nium chloride (ADBAC). Consequently, commercial formulations containing one or more of these additional active ingredients were selected, in addition to one formulation containing only DDAC as an active ingredient. The selected commercial formulations tended to reduce skin penetration of DDAC. This was most pronounced with the formulation containing the highest concentration of formaldehyde (196 mg/mL) and glutaraldehyde (106 mg/mL), which reduced the flux of DDAC across the skin by 95%. The reduction caused by the only tested formulation containing no other active ingredients than DDAC, and thus incorporating no aldehydes, was smallest, and did not reach statistical significance. I n chapter 6 of this thesis, a simple in silico model to predict finite dose dermal absorption from infinite dose data (kp and lag time) and the stratum corneum/water partition coefficient (KSC,W) was developed. This model was tentatively called Dermal Absorption Model for Extrapolation (DAME). As dermal exposure may occur under a large variety of conditions leading to quite different rates of absorption, such a predictive model using simple experimental or physicochemical inputs provides a cost-effective means to estimate dermal absorption under different conditions. To evaluate the DAME, a series of in vitro dermal absorption experiments was performed under both infinite and finite dose conditions using a variety of different substances. The kp’s and lag times determined in the infinite dose experiments were entered into DAME to predict relative dermal absorption value under finite dose conditions. For six substances, the predicted relative dermal absorption under finite dose conditions was not statistically different from the measured value. For all other substances, measured absorption was overpredicted by DAME, but most of the overpredicted values were still lower than 100%, the European default absorption value for the tested compounds. In conclusion, our finite dose prediction model (DAME) provides a useful and cost-effective estimate of in vitro dermal absorption, to be used in risk assessment for non-volatile substances dissolved in water at non-irritating concentrations. I n chapter 7 of this thesis, the results of the research reported in chapters 2 to 6 were put into perspective, the pitfalls and promises emanating from them discussed and general conclusions drawn. The possible influence of vehicles on absorption and the possible impact of irritative or corrosive vehicles or chemicals on the skin barrier have been demonstrated in this thesis. An in silico predictive model tentatively called DAME was developed, which enables the user to evaluate a variety of dermal exposure scenarios with limited experimental data (kp and lag time) and easy to obtain physicochemical properties (MW and log KOW). The predictions of our experiments reported in chapter 6 were compared to those of the Finite Dose Skin Permeation (FDSP) model published on the internet by the US Centers for Disease Control and Prevention (CDC). DAME outperformed FDSP (R2 of the correlation predicted/measured potential absorption 0.64 and 0.12, respectively). At present, the applicability domain of DAME is limited to non-volatile substances dissolved in aqueous solvents. However, in future the model will be adapted to include volatile substances as well. Altogether, it is concluded that dermal exposure can be an important factor in risks posed by chemicals and should be taken into account in risk assessment. The methods to actually do this are still open for further improvement to better account for the various factors influencing skin penetration and to develop adequate combinations of in vitro and in silico models that can accurately predict human dermal absorption.

Background Measurement error in exposure variables is an important issue in epidemiological studies that relate exposures to health outcomes. Such studies, however, usually pay limited attention to the quantitative effects of exposure measurement error on estimated exposure-outcome associations. Therefore, the estimators for exposure-outcome associations are prone to bias. Existing methods to adjust for the bias in the associations require a validation study with multiple replicates of a reference measurement. Validation studies with multiple replicates are quite costly and therefore, in some cases only a single–replicate validation study is conducted besides the main study. For a study that does not include an internal validation study, the challenge in dealing with exposure measurement error is even bigger. The challenge is how to use external data from other similar validation studies to adjust for the bias in the exposure-outcome association. In accelerometry research, various accelerometer models have currently been developed. However, some of these new accelerometer models have not been properly validated in field situations. Despite the widely recognized measurement error in the accelerometer, some accelerometers have been used to validate other instruments, such as physical activity questionnaires, in measuring physical activity. Consequently, if an instrument is validated against the accelerometer, and the accelerometer itself has considerable measurement error, the observed validity in the instrument being validated will misrepresent the true validity. Methodology In this thesis, we adapted regression calibration to adjust for exposure measurement error for a single-replicate validation study with zero-inflated reference measurements and assessed the adequacy of the adapted method in a simulation study. For the case where there is no internal validation study, we showed how to combine external data on validity for self-report instruments with the observed questionnaire data to adjust for the bias in the associations caused by measurement error in correlated exposures. In the last part, we applied a measurement error model to assess the measurement error in physical activity as measured by an accelerometer in free-living individuals in a recently concluded validation study. Results The performance of the proposed two-part model was sensitive to the form of continuous independent variables and was minimally influenced by the correlation between the probability of a non-zero response and the actual non-zero response values. Reducing the number of covariates in the model seemed beneficial, but was not critical in large-sample studies. We showed that if the confounder is strongly linked with the outcome, measurement error in the confounder can be more influential than measurement error in the exposure in causing the bias in the exposure-outcome association, and that the bias can be in any direction. We further showed that when accelerometers are used to monitor the level of physical activity in free-living individuals, the mean level of physical activity would be underestimated, the associations between physical activity and health outcomes would be biased, and there would be loss of statistical power to detect associations. Conclusion The following remarks were made from the work in this thesis. First, when only a single-replicate validation study with zero-inflated reference measurements is available, a correctly specified regression calibration can be used to adjust for the bias in the exposure-outcome associations. The performance of the proposed calibration model is influenced more by the assumption made on the form of the continuous covariates than the form of the response distribution. Second, in the absence of an internal validation study, carefully extracted validation data that is transportable to the main study can be used to adjust for the bias in the associations. The proposed method is also useful in conducting sensitivity analyses on the effect of measurement errors. Lastly, when “reference” instruments are themselves marred by substantial bias, the effect of measurement error in an instrument being validated can be seriously underestimated.

Exposure models are used to calculate the amount of potential harmful chemicals ingested by a human population. Examples of harmful chemicals are residues of pesticides, chemicals entering food from the environment (such as dioxins, cadmium, lead, mercury), and chemicals that are generated via heating (such as acrylamide and furans). In this report we describe the characteristics of two types of models: the first for calculating the short term-intake, and the second for calculating long-term intake. These models currently result in the most realistic estimation of chemical intake via food.

Exposure models are used to calculate the amount of potential harmful chemicals ingested by a human population. Examples of harmful chemicals are residues of pesticides, chemicals entering food from the environment (such as dioxins, cadmium, lead, mercury), and chemicals that are generated via heating (such as acrylamide and furans). In this report we describe the characteristics of two types of models: the first for calculating the short term-intake, and the second for calculating long-term intake. These models currently result in the most realistic estimation of chemical intake via food.

Background: Risk assessment can potentially be improved by closely linked experiments in the disciplines of epidemiology and toxicology. This was explored for isoflavones in a case study. For isoflavones potential beneficial health effects have been suggested, but discussions on their safety are ongoing as well. Aims and methods: Effects of isoflavone supplements on gene expression were studied in white blood cells (PBMCs) and adipose tissue, among postmenopausal women in two human intervention studies. To advance risk assessment, the dose response relation between intake and blood levels was studied with a log-linear regression model as well as the comparability of the human gene expression profiles with results from a rat experiment using multivariate analysis. Results: In both PBMCs and adipose tissue, changes in gene expression profiles pointed at effects of isoflavones on energy metabolism, inflammation and cell cycle; these effects were modified by supplement composition and equol-producing phenotype. Hypothesized estrogen-responsive effects were not observed. For the intake range of 0-100mg/day, the plasma concentrations of daidzein, equol, genistein and total isoflavones were quantified, interindividual variation. Expression of estrogen-responsive gene profiles and other biological pathways could be quantitatively compared between PBMCs and adipose tissue, as well as between humans and rats. en nog iets Conclusion: Effects of isoflavone supplementation on gene expression in PBMCs and adipose tissue of postmenopausal women suggest mainly beneficial effects of a dose of ~100mg/day. The absence of a clear estrogen-like response suggested a limited role of the estrogen receptor in isoflavone induced gene expression in postmenopausal women. The trials and quantitative models provide important tools[AG1] that enable further exploration of intertissue and interspecies comparability and advancing the use of transcriptomics in assessing risks and benefits. Modelling data from human and animal studies provide important possibilities for further exploration of intertissue and interspecies similarities and for the use of transcriptomics in improving risk assessment.

Background: Risk assessment can potentially be improved by closely linked experiments in the disciplines of epidemiology and toxicology. This was explored for isoflavones in a case study. For isoflavones potential beneficial health effects have been suggested, but discussions on their safety are ongoing as well. Aims and methods: Effects of isoflavone supplements on gene expression were studied in white blood cells (PBMCs) and adipose tissue, among postmenopausal women in two human intervention studies. To advance risk assessment, the dose response relation between intake and blood levels was studied with a log-linear regression model as well as the comparability of the human gene expression profiles with results from a rat experiment using multivariate analysis. Results: In both PBMCs and adipose tissue, changes in gene expression profiles pointed at effects of isoflavones on energy metabolism, inflammation and cell cycle; these effects were modified by supplement composition and equol-producing phenotype. Hypothesized estrogen-responsive effects were not observed. For the intake range of 0-100mg/day, the plasma concentrations of daidzein, equol, genistein and total isoflavones were quantified, interindividual variation. Expression of estrogen-responsive gene profiles and other biological pathways could be quantitatively compared between PBMCs and adipose tissue, as well as between humans and rats. en nog iets Conclusion: Effects of isoflavone supplementation on gene expression in PBMCs and adipose tissue of postmenopausal women suggest mainly beneficial effects of a dose of ~100mg/day. The absence of a clear estrogen-like response suggested a limited role of the estrogen receptor in isoflavone induced gene expression in postmenopausal women. The trials and quantitative models provide important tools[AG1] that enable further exploration of intertissue and interspecies comparability and advancing the use of transcriptomics in assessing risks and benefits. Modelling data from human and animal studies provide important possibilities for further exploration of intertissue and interspecies similarities and for the use of transcriptomics in improving risk assessment.

The genus Cronobacter consists of Gram-negative, motile, non-spore forming, facultative anaerobic bacteria, and was originally defined as one species “Enterobacter sakazakii” within the genus Enterobacter in 1980. Cronobacter spp. have been documented as a rare cause of outbreaks and sporadic cases of neonatal meningitis, necrotizing enterocolitis, and sepsis in infants with a high mortality. Among these infants, those at greatest risk are infants less than 2 months of age, particularly pre-term infants, low birth weight (LBW) infants (< 2500 g), and immuno-compromised infants. At the onset of the work for this thesis, Cronobacter spp. had been isolated from milk-based powdered formulae which have a direct link to the sub-population at greatest risk. However, there was a need to more closely investigate whether and where Cronobacter spp. occurs in environments in which these powdered are manufactured and packed but also to investigate other sources which could lead to exposure of vulnerable sub-populations. The main objective of this study was to develop isolation and detection methods that would allow quick and reliable investigation into the occurrence of the micro-organism in potential sources. Furthermore, more insight into the growth behavior of Cronobacter spp. in reconstituted infant formula was necessary in order to provide data to be used in Microbiological Risk Assessment (MRA) dedicated to this particular food product. A selective enrichment method was developed for the rapid and reliable enrichment and detection of Cronobacter spp. in environmental samples. The detection method which was developed is based on two features of Cronobacter spp. combined: their yellow pigmented colonies when grown on tryptone soy agar and their constitutive -glucosidase, which can be detected in a 4-h colorimetric assay. The initially developed method and refinements thereof were applied for routine screening for the presence of Cronobacter spp. in environmental samples and a variety of food products manufactured or marketed in The Netherlands. The detection method described in this thesis has been the basis for a series of media for Cronobacter spp. that have recently been commercialized. Quantitative data on product contamination at manufacture, during preparation, and also growth after reconstitution are required in order to assess the risk associated with Cronobacter spp. exposure. Next to that, tools are needed to asses the micro-organisms growth potential as well as its inactivation (thus, its survival) due to specific control measures applied. In this thesis, predictive growth models were developed that capture key growth parameters. Minimum– and maximum temperatures estimated with the Secondary Rosso equation were 3.6 ºC and 47.6 ºC, respectively. The estimated lag time of the micro-organisms was found to vary from 83.3 ± 18.7 h at 10 ºC to 1.73 ± 0.43 h at 37 ºC and could be described with the hyperbolic model and reciprocal square root relation. The models for growth rates and lag times as a function of temperatures obtained during this study allow estimating the potential growth of Cronobacter spp. in reconstituted infant formula stored at any temperature below 47 C. As growth rates of Cronobacter at refrigeration temperatures are relatively small, caregivers are advised to store reconstituted infant at low temperature as a control measure to prevent microbial growth. It is evident that storage of reconstituted formula in a refrigerator may require a significant amount of the time before the formula reach the targeted refrigeration temperature. Therefore, a mathematical model was built to predict the temperature profile and the resulting growth of Cronobacter spp. during cooling, i.e. under dynamic temperature conditions. Predictions showed that proliferation of Cronobacter spp. during cooling strongly depends on the size of the container used for storage and that it may be prevented by limiting the volume to be cooled to portion-size only or by reconstituting at temperatures of 25 °C or lower. The survival of two Cronobacter strains in dry powdered infant formula (PIF) was tested and compared to the survival of six other bacterial strains after inoculation and storage at several temperatures between 7 and 42 ºC. The effect of temperature on survival in PIF, was described using both the Weibull distribution model and the log-linear model. Differences were found in the rate of survival that can be due to difference in the resistance to inactivation in dry environments between Cronobacter species, which could be relevant to consider when establishing quantitative risk assessments on consumer risks related to PIF. The research described in this thesis contributes to the existing knowledge on the natural habitat of Cronobacter spp. and its occurrence and behavior in PIF. The models developed for quantifying the growth of Cronobacter spp. in reconstituted formulae under various conditions can be applied in risk assessments set-up to estimate the probability of vulnerable sub-populations becoming ill after consuming infant formulae. International and national governmental bodies may use these predictive models in risk assessments and to establish guidelines for health care professionals to provide effective hygiene training to parents and professional caregivers to ensure that PIF is prepared handled and stored appropriately.

The genus Cronobacter consists of Gram-negative, motile, non-spore forming, facultative anaerobic bacteria, and was originally defined as one species “Enterobacter sakazakii” within the genus Enterobacter in 1980. Cronobacter spp. have been documented as a rare cause of outbreaks and sporadic cases of neonatal meningitis, necrotizing enterocolitis, and sepsis in infants with a high mortality. Among these infants, those at greatest risk are infants less than 2 months of age, particularly pre-term infants, low birth weight (LBW) infants (< 2500 g), and immuno-compromised infants. At the onset of the work for this thesis, Cronobacter spp. had been isolated from milk-based powdered formulae which have a direct link to the sub-population at greatest risk. However, there was a need to more closely investigate whether and where Cronobacter spp. occurs in environments in which these powdered are manufactured and packed but also to investigate other sources which could lead to exposure of vulnerable sub-populations. The main objective of this study was to develop isolation and detection methods that would allow quick and reliable investigation into the occurrence of the micro-organism in potential sources. Furthermore, more insight into the growth behavior of Cronobacter spp. in reconstituted infant formula was necessary in order to provide data to be used in Microbiological Risk Assessment (MRA) dedicated to this particular food product. A selective enrichment method was developed for the rapid and reliable enrichment and detection of Cronobacter spp. in environmental samples. The detection method which was developed is based on two features of Cronobacter spp. combined: their yellow pigmented colonies when grown on tryptone soy agar and their constitutive -glucosidase, which can be detected in a 4-h colorimetric assay. The initially developed method and refinements thereof were applied for routine screening for the presence of Cronobacter spp. in environmental samples and a variety of food products manufactured or marketed in The Netherlands. The detection method described in this thesis has been the basis for a series of media for Cronobacter spp. that have recently been commercialized. Quantitative data on product contamination at manufacture, during preparation, and also growth after reconstitution are required in order to assess the risk associated with Cronobacter spp. exposure. Next to that, tools are needed to asses the micro-organisms growth potential as well as its inactivation (thus, its survival) due to specific control measures applied. In this thesis, predictive growth models were developed that capture key growth parameters. Minimum– and maximum temperatures estimated with the Secondary Rosso equation were 3.6 ºC and 47.6 ºC, respectively. The estimated lag time of the micro-organisms was found to vary from 83.3 ± 18.7 h at 10 ºC to 1.73 ± 0.43 h at 37 ºC and could be described with the hyperbolic model and reciprocal square root relation. The models for growth rates and lag times as a function of temperatures obtained during this study allow estimating the potential growth of Cronobacter spp. in reconstituted infant formula stored at any temperature below 47 C. As growth rates of Cronobacter at refrigeration temperatures are relatively small, caregivers are advised to store reconstituted infant at low temperature as a control measure to prevent microbial growth. It is evident that storage of reconstituted formula in a refrigerator may require a significant amount of the time before the formula reach the targeted refrigeration temperature. Therefore, a mathematical model was built to predict the temperature profile and the resulting growth of Cronobacter spp. during cooling, i.e. under dynamic temperature conditions. Predictions showed that proliferation of Cronobacter spp. during cooling strongly depends on the size of the container used for storage and that it may be prevented by limiting the volume to be cooled to portion-size only or by reconstituting at temperatures of 25 °C or lower. The survival of two Cronobacter strains in dry powdered infant formula (PIF) was tested and compared to the survival of six other bacterial strains after inoculation and storage at several temperatures between 7 and 42 ºC. The effect of temperature on survival in PIF, was described using both the Weibull distribution model and the log-linear model. Differences were found in the rate of survival that can be due to difference in the resistance to inactivation in dry environments between Cronobacter species, which could be relevant to consider when establishing quantitative risk assessments on consumer risks related to PIF. The research described in this thesis contributes to the existing knowledge on the natural habitat of Cronobacter spp. and its occurrence and behavior in PIF. The models developed for quantifying the growth of Cronobacter spp. in reconstituted formulae under various conditions can be applied in risk assessments set-up to estimate the probability of vulnerable sub-populations becoming ill after consuming infant formulae. International and national governmental bodies may use these predictive models in risk assessments and to establish guidelines for health care professionals to provide effective hygiene training to parents and professional caregivers to ensure that PIF is prepared handled and stored appropriately.

Het RIVM en het RIKILT-Instituut voor Voedselveiligheid schatten hoeveel chemische stoffen mensen via voeding binnenkrijgen. De huidige methode zou meer inzicht moeten kunnen geven in de mate waarin deze blootstellingsschattingen onzekerheden bevatten. Het kwantificeren van onzekerheden sluit aan bij de internationale wetenschappelijke activiteiten die momenteel op het gebied van onzekerheidsanalyse van blootstellingsschattingen gaande zijn. Naast het kwantificeren van meer onzekerheden moet de invloed van de (overgebleven) niet-gekwantificeerde onzekerheden op de blootstellingsschatting worden beschouwd

Het RIVM en het RIKILT-Instituut voor Voedselveiligheid schatten hoeveel chemische stoffen mensen via voeding binnenkrijgen. De huidige methode zou meer inzicht moeten kunnen geven in de mate waarin deze blootstellingsschattingen onzekerheden bevatten. Het kwantificeren van onzekerheden sluit aan bij de internationale wetenschappelijke activiteiten die momenteel op het gebied van onzekerheidsanalyse van blootstellingsschattingen gaande zijn. Naast het kwantificeren van meer onzekerheden moet de invloed van de (overgebleven) niet-gekwantificeerde onzekerheden op de blootstellingsschatting worden beschouwd

The consumption of food contaminated with the bacterium Bacillus cereus may lead to either symptoms of vomiting or symptoms of diarrhoea. As the symptoms are rather mild, few patients seek medical attention. Therefore, it is hard to estimate the number of cases. To improve estimation of this number and to gather more insight into factors that may determine the chance for disease, the following aspects have been investigated: the prevalence of B. cereus in food commodities in the Netherlands, the survival of B. cereus in simulated gastric fluid and the growth potential in simulated intestinal fluid, the interaction between B. cereus and intestinal cells, and the production of toxins that cause the disease symptoms. Together with literature data the results of these investigations have been used to determine the chance for disease by B. cereus. In combination with the number of meals that are consumed and that are contaminated with B. cereus, the yearly number of disease cases in the Netherlands has been estimated using different scenarios.

This report describes the result of a study into the exposure to harmful compounds via more than one source / route (so-called aggregate exposure) in relation to the relevance of extending dietary exposure assessments as performed using the Monte Carlo Risk Assessment (MCRA) programme with other sources than diet (and drinking water) or other routes. An overview is given of relevant groups of compounds for which aggregate exposure may be relevant, and of models available to calculate aggregate exposure, including NORMTOX and several American models. Also relevant stakeholders of aggregate exposure are identified.

This report describes the result of a study into the exposure to harmful compounds via more than one source / route (so-called aggregate exposure) in relation to the relevance of extending dietary exposure assessments as performed using the Monte Carlo Risk Assessment (MCRA) programme with other sources than diet (and drinking water) or other routes. An overview is given of relevant groups of compounds for which aggregate exposure may be relevant, and of models available to calculate aggregate exposure, including NORMTOX and several American models. Also relevant stakeholders of aggregate exposure are identified.