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A major hurdle in drug development is the limited benefit of efficacy data in animals for use in the clinic. For each patient (sub)group or disease, many different animal models exist to provide insight into drug efficacy. However, it is often unclear how predictive the outcomes of drugs tested in these models are for the clinic, and on what basis researchers select a specific model. The main goal of this thesis is to gain insight into the selection of animal models for drug efficacy. We studied the extent of the low value of animal models for predicting efficacy. Then, we analyzed underlying causes of this low value. To this end, we studied the role of different stakeholders in the selection process, such as researchers, local and national ethics committees, and funding agencies. Finally, we present an instrument that supports the evaluation of similarities between animal models and patients: the Framework to Identify Models of Disease (FIMD). FIMD provides a solution to assess the value of animal models for efficacy. We applied FIMD to validate two animal models of cow's milk allergy in young children. We demonstrated that the instrument contributes to validating animal models for drug efficacy and thus can facilitate appropriate animal model selection. When applied, this instrument provides a scientific basis for policy recommendations and changes, which can lead to a more responsible use of animals in drug development.

A major hurdle in drug development is the limited benefit of efficacy data in animals for use in the clinic. For each patient (sub)group or disease, many different animal models exist to provide insight into drug efficacy. However, it is often unclear how predictive the outcomes of drugs tested in these models are for the clinic, and on what basis researchers select a specific model. The main goal of this thesis is to gain insight into the selection of animal models for drug efficacy. We studied the extent of the low value of animal models for predicting efficacy. Then, we analyzed underlying causes of this low value. To this end, we studied the role of different stakeholders in the selection process, such as researchers, local and national ethics committees, and funding agencies. Finally, we present an instrument that supports the evaluation of similarities between animal models and patients: the Framework to Identify Models of Disease (FIMD). FIMD provides a solution to assess the value of animal models for efficacy. We applied FIMD to validate two animal models of cow's milk allergy in young children. We demonstrated that the instrument contributes to validating animal models for drug efficacy and thus can facilitate appropriate animal model selection. When applied, this instrument provides a scientific basis for policy recommendations and changes, which can lead to a more responsible use of animals in drug development.

A major hurdle in drug development is the limited benefit of efficacy data in animals for use in the clinic. For each patient (sub)group or disease, many different animal models exist to provide insight into drug efficacy. However, it is often unclear how predictive the outcomes of drugs tested in these models are for the clinic, and on what basis researchers select a specific model. The main goal of this thesis is to gain insight into the selection of animal models for drug efficacy. We studied the extent of the low value of animal models for predicting efficacy. Then, we analyzed underlying causes of this low value. To this end, we studied the role of different stakeholders in the selection process, such as researchers, local and national ethics committees, and funding agencies. Finally, we present an instrument that supports the evaluation of similarities between animal models and patients: the Framework to Identify Models of Disease (FIMD). FIMD provides a solution to assess the value of animal models for efficacy. We applied FIMD to validate two animal models of cow's milk allergy in young children. We demonstrated that the instrument contributes to validating animal models for drug efficacy and thus can facilitate appropriate animal model selection. When applied, this instrument provides a scientific basis for policy recommendations and changes, which can lead to a more responsible use of animals in drug development.

In a study in which male rats have been exposed to 0,20, 60, 200, 600 and 2000 mg di(isononyl)phthalate (DINP)/kg diet for 2 weeks, body weight and liver weight and a number of enzyme parameters (palmitoyl coenzyme-A oxidase (PCO), enoyl coenzyme-A hydratase (ECH), carnitine acetyl transferase (CAT) and lauric acid hydroxylase (LAH)) have been determined in liver homogenates which are related with peroxisome proliferation. No effects have been observed on both liver and body weight. PCO, CAT and LAH, showed no effect at 200 mg DINP/kg diet, corresponding with 18.2 mg/kg b.w./day. ECH appeared to be the most sensitive parameter with a dose-without-effect of 60 mg DINP/kg diet, corresponding to 5.7 mg DINP/kg b.w./day. This value of 60 mg/kg diet has been defined as overall no-observed-effect-level for DINP in this experiment.

In a subchronic toxicity study 4 groups of 10 Sprague-Dawley rats/group/sex received 0, 5, 20 and 80 mg ergotamine tartrate (EAT)/kg diet respectively during 13 weeks. Food intake and water intake were measured twice a week. Body weight gain was recorded weekly. After 7 weeks and 12 weeks urine and serum were sampled to measure the kidney function. After 10 weeks blood was sampled for haematological examination. At the end of the experiment blood and a small proportion of the liver were sampled for biochemical analyses. After necropsy organ weights were recorded and tissues were fixed for a complete histopathological examination. In female rats the body weight gain, and food intake were decreased at a dose level of 80 mg EAT/kg diet. The haematological examinations revealed increased RBC and PCV values in the animals of the high dose group (80 mg EAT/kg diet) only. Biochemical examination showed increased alkaline phosphatase activity, LDL-cholesterol and decreased glucose concentrations in the serum of animals of the high dose group. Urine-analyses revealed increased urine volume only in the females of the high dose group. Relative spleen and brain weights in the high dose group were higher than in the control and pituitary weight was lower in the high dose group. Macroscopical examination revealed pale thyroids and slight dilation of the uterus, haemorrhagic changes in the lymph nodes in animals of the high dose group. The only treatment-related finding in histopathology was the muscular atrophy in the caudal longitudinal muscles of the tail of animals of the high dose group. The no-toxic effect level in this experiment was 20 mg EAT/kg diet equivalent 0.9 mg ergotamine/kg bw.

In a study in which male rats have been exposed to 0, 20, 60, 200, 600 and 2000 mg di(isodecyl)phthalate (DIDP)kg diet for 2 weeks, body weight and liver weight and a number of enzyme parameters (palmitoyl coenzyme-A oxidase (PCO), enoyl coenzyme-A hydratase (ECH), carnitine acetyl transferase (CAT) and lauric acid hydroxylase (LAH) have been determined in liver homogenates which are related with peroxisome proliferation. No effects have been observed on liver and body weight. CAT appeared to be the most sensitive parameter with a dose-without-effect (DWE) of 60 mg DIDP/kg diet, corresponding with 5.6 mg DIDP/kg b.w./day. The other enzyme parameters PCO, ECH and LAH showed no effect at 200 mg DIDP/kg diet, corresponding with 18.4 mg/kg b.w/day. Since CAT is considered to be not specific for peroxisome proliferation this latter value has been established as overall no-observed-effect-level.

Acute blootstelling aan ozon, bijvoorbeeld gedurende een zomersmogepisode van enkele dagen, resulteert in een verscheidenheid aan effecten welke zowel in onderzoek bij proefdieren als mensen zijn waargenomen. Over de aard en het tijdsverloop van de inflammatoire respons en het herstelproces na blootstelling is vooral veel bekend uit proefdieronderzoek. Bij de mens zijn het geforceerd uitademingsvolume in 1 seconde (FEV 1) en de maximale expiratoire flow of piefflow (PF) veelvuldig gebruikte longfunctieparameters. Dit dierexperimenteel onderzoek is uitgevoerd om de invloed van het aantal expositiedagen op het effect van ozonblootstelling op proefdieren te kunnen beschrijven. Ratten werden 's nachts gedurende 1, 2 of 3 dagen 12 uur achtereen blootgesteld aan 800 mug/m3 ozon. Het verloop van longfunctieveranderingen tijdens deze episodische blootstelling werd onderzocht met de methode van de geforceerde uitademing. De Maximale Expiratoire Flow Volume - curve (MEFV) werd gemeten direct na blootstelling. Herstel werd onderzocht door longfunctiemetingen uit te voeren op 24 en 48 uur na afloop van de blootstelling. Na de eerste blootstelling werden duidelijke veranderingen in de MEFV-curve gemeten, welke na een herstelperiode van 24 uur waren verdwenen. Na de tweede blootstelling werden veranderingen van dezelfde grootte waargenomen. Ook hier waren de gemeten veranderingen na 24 uur herstel weer verdwenen. Echter, direct na de derde blootstelling zijn geen veranderingen meer waarneembaar. Longfunctieveranderingen tijdens een 3 dagen durende episodische blootstelling zijn na de eerste en tweede blootstellingsdag waarneembaar. Na de derde dag is geen effect meer meetbaar. Dit verloop komt overeen met het verloop van de inflammatoire respons gemeten in longspoelvloeistof bij onderzoek met ratten met eenzelfde blootstellingsprotocol. Bij onderzoek met vrijwilligers is eveneens een dergelijk tijdsverloop in longfunctieveranderingen en ontstekingsrespons vastgesteld.

Studies in de mens laten een afname van de longfunctierespons zien voor ozon met het ouder worden, ofschoon de symptomenincidentie dezelfde blijft. Het beperkt aantal experimentele dierstudies laat een diffuse respons zien zonder een duidelijke relatie met leeftijd. Deze data suggereren dus dat jonge dieren niet gevoeliger zouden zijn dan volwassen dieren. Beschikbare toxiciteitsdata bij mens en dier hebben niet afdoende aangetoond dat er geslachtsverschillen bestaan ten aanzien van de gevoeligheid voor ozon. Deze dierstudie onderzocht mogelijke leeftijds- en geslachts- afhankelijke verschillen van de respons van de long op een eenmalige en herhaalde blootstelling aan ozon. Ratten van verschillende leeftijdsgroepen (1, 3, 9 en 18 maanden, overeenkomend met gespeende, jong volwassen, volwassen en relatief oude dieren) werden acuut blootgesteld aan 800 mug/m3 ozon gedurende een dag (12h) of herhaaldelijk blootgesteld (7 dagen, 12 h/d). Verscheidene parameters gerelateerd aan longfunctie, ontsteking en weefselschade, werden gehanteerd voor evaluatie van de ozongevoeligheid. Longfunctietesten lieten geen leeftijdsafhankelijke, noch ozoneffecten zien. Er werden geen leeftijdsafhankelijke effecten gezien in de volume gecorrigeerde compliance van controle dieren. Waarschijnlijk werd door het experimentele regime het niet optimale meettijdstip gekozen om een maximale respons te meten. Zowel morfologisch als morfometrisch werden leeftijdsafhankelijke effecten van ozonblootstelling in de longen waargenomen. Vanaf de leeftijd van 3 maanden bleken de dieren statistisch significant minder gevoeliger te worden voor ozon. Met betrekking tot de biochemische parameters bleek de basale antioxidant capaciteit in de longen van controle dieren vanaf de leeftijd van 3 maanden af te nemen. Toch stegen de enzymactiviteiten in de longen bij dieren van 9 en 18 maanden na ozonblootstelling. Er was geen statistisch significant leeftijdsafhankelijk effect van ozon. De afname van de percentuele toename van het eiwit- en albuminegehalte in longspoelvloeistof (een maat voor capillaire-alveolaire permeabiliteit) in aan ozonblootgestelde ratten vanaf 1 maand veronderstelt een afname in gevoeligheid met toenemende leeftijd. De geleidelijke afname van het netto percentage polymorfkernigen in de longspoelvloeistof na ozonblootstelling in mannelijke dieren naar mate de dieren ouder zijn, versterkt de veronderstelling van een afnemende gevoeligheid in oude ratten. Het resultaat van Listeria infectie proeven liet een bevestiging zien van vorige studies m.b.t. een statistisch significant ozoneffect. Doch er was geen statistisch significant verschil tussen de verschillende leeftijdgroepen in de weerstand tegen een Listeria infectie na ozonblootstelling. Concluderend blijkt dat enkele parameters zoals weefselschade en longonsteking duiden op een toegenomen reactie in jonge dieren. Andere parameters zoals weerstand tegen een Listeria infectie, longfunctie en antioxidant enzym respons wijzen op geen onderlinge verschillen tussen de leeftijdsgroepen in hun gevoeligheid voor ozon. Er werden bij alle onderzochte parameters geen geslachts- gerelateerde verschillen waargenomen ten aanzien van de respons op ozon. Eventuele verschillen van de respons op ozon van de diverse leeftijdsgroepen zouden verklaard kunnen worden door de biologische gevoeligheid voor ozon van de target-plaats, of door de verschillen in longdosimetrie bij dieren van verschillende leeftijden. De resultaten van deze kortdurende ozonblootstellingen zijn moeilijk te interpreteren voor wat betreft de leeftijd-gerelateerde gezondheidseffecten.

The toxicokinetics of [3H]-alpha-solanine after oral (po) and intravenous (iv) administration in rats and hamsters were studied, in order to decide which is the most appropriate model in risk assessment studies. The iv dose was 54 mug/kg; the oral dose was 170 mug/kg. After iv administration, the toxicokinetics of total radioactivity in blood were comparable in rats and hamsters. However, the clearance of total radioactivity from plasma was more effective in rats than in hamsters The half-lives of distribution and of the terminal phase of unchanged alpha-solanine were not different between rats and hamsters, whereas the systemic and the metabolic clearance were about 1.6 and 2.7 times higher in rats than in hamsters, respectively. The clearance of unchanged alpha-solanine is more effective than that of total radioactivity. After oral administration in rats and hamsters, the mean bioavailability of total radioactivity is about 29% and 57%, respectively, whereas the bioavailability of unchanged alpha-solanine is only 1.6% and 3.2%, respectively, when compared with iv administration. T1/2 el of alpha-solanine after oral administration was in rats a factor of four and in hamsters a factor of two shorter than after iv administration. A strong retention of radioactivity was seen in the hamsters after oral administration; only 40% of the dose was excreted within seven days vs 90% in rats. In some hamsters, severe damage of the duodenal wall was observed after oral administration. Based on these data, it was decided that the hamster is a more appropriate model in (sub)-chronic toxicity studies with alpha-solanine than the rat.

Two animal experiments are described in which male rats have been exposed to di(2-ethylhexyl)phthalate (DEHP) for 2 and 4 weeks. Besides morphometric analysis using both light and electron microscopy, a number of enzyme parameters in liver homogenates have been determined which have a relation with the proliferation of peroxisomes in hepatocytes, such as palmitoyl coenzyme-A oxidase (PCO), enoyl coenzyme-A hydratase (ECH), carnitine acetyl transferase (CAT), catalase (cat) and lauric acid hydroxylase (LAH). Glutamate dehydrogenase (GIDH) was measured as a control enzyme for mitochondria. For all enzymes, except GIDH, a dose-response relationship was observed. There were not many differences between the results of the parameters examined in the 2 and 4 weeks exposed animals. In the 2 weeks exposure experiment, the enzyme parameters appeared to be slightly more sensitive. Therefore the parameters from this experiment were taken for determination of the doses-without-effect (DWE). Also the morphometric analysis has been performed on the 2 weeks-experiment. Although CAT turned out the most sensitive parameter, this enzyme activity was not considered in the establishment of an overall no-observed-effect-level since it is not specific for peroxisome proliferation. The second most sensitive parameters were the morphometric analysis and the induction of LAH and ECH activity (DWE = 5 mg/kg b.w./day) followed by PCO (DWE = 18 mg/kg b.w./day). Catalase activity was not a very sensitive parameter (52 mg/kg b.w./day). An overall no-observed-effect-level for DEHP was established as 5 mg/kg b.w./day, based on the parameters mentioned for peroxisome proliferation.

Morphometric analysis was performed on liver sections of rats at light (LM) and electron microscopical (EM) level to demonstrate proliferation of peroxisomes after administration of di (2-ethylhexyl)phthalate at dietary levels of 0, 60, 200, 600, 2000 and 6000 mg/kg diet for 2 weeks. Enzyme histochemical demonstration of catalase was carried out to demonstrate the presence of peroxisomes. The results of the LM-morphometric analysis will be compared with those of the biochemical analysis, reported elsewhere. The LM-morphometric data will be tested by EM-morphometry in a limited number of groups. With the LM- as well as the EM-method the volume density of peroxisomes and the number of peroxisomes were significantly increased after administration of 200 mg DEHP/kg diet during 2 weeks. However, LM-morphometry of peroxisomes by enzyme histochemical demonstration of catalase is suitable to compare the capacity of various phthalate esters to induce proliferation of peroxisomes, realising that the measured values of volume density at LM-level are not true values, but relative values. With the catalase reaction, enzyme histochemical morphometry of peroxisomes showed a dose-without-effect of 60 mg DEHP/kg diet.

This report describes the realisation and implementation of working procedures in compliance with Good Laboratory Practice, as performed by the Central Animal Facility of Division II Pharmacology and Toxicology. This report froms a basis for a quality system with the pivot aim to meet Sterlab requirements.

In this report a rodent Physiologically Based PharmacoKinetic (PBPK) model for 2,3,7,8-tetrachlorodibenzodioxin is described. Validation studies, in which model simulations of TCDD disposition were compared with in vivo TCDD disposition in rodents exposed to TCDD, showed that the model adequately predicted the in vivo toxicokinetics of TCDD across species (mouse, rat), dose-schedule (acute semi-chronic chronic) and route of administration (oral subcutaneous). It was concluded that PBPK models are suited to establish quantitative relationships between the external dose of TCDD (amount of TCDD administered per kg body weight) and the internal dose (concentration of TCDD at the organ level). The relationship between internal dose and the resulting organ toxicity will be elaborated in subsequent reports. The results will be used in a quantitative risk assessment of the human exposure situation, in particular the exposure of suckling infants to dioxins from mother's milk.

A subchronic toxicity experiment with propyl gallate in SPF-derived Wistar RIVM:TOX rats was performed. Groups of 10 female and 10 male rats were fed a semisynthetic diet containing 0, 490, 1910 and 7455 mg propyl gallate /kg feed. Body weight gain was recorded weekly and food-intake twice weekly. Other parameters comprised haematology, biochemical determinations in urine, serum and liver and complete histopathological examinations. Adverse effects of propyl gallate observed in the high dose group were effects on the haemopoeitic system reflected in the haematological parameters and the morphological changes in the spleen. The other effects observed comprised decreased incidence of the nephrocalcinosis in female rats, the increased activity of EROD in the high dose group and increased activity of the conjugating enzymes; glucorynyl-transferase and glutathion-s-transferase, in the mid and high dose group of propyl gallate. The effects on the nephrocalcinosis and on the conjugating enzymes may be considered as not adverse. The no observed adverse effect level (NOAEL) is 1910 mg propyl gallate /kg feed corresponding with 135 mg propyl gallate /kg body weight.

Vier typen adjuvantia zijn geevalueerd als alternatief voor het gebruik van Freund's compleet adjuvant in muizen. De volgende adjuvantia zijn geevalueerd: een water-in-olie emulsie (Specol), een micro-organisme (Lactobacillus), een immuunstimulerend complex met ingebouwd glycoproteine van rabies virus (RV-iscoms) en een saponine. Quil A. De adjuvantia en controle groepen (fysiologisch zout) zijn gecombineerd met drie zwak immunogene antigenen (een synthetisch antigeen, een autoantigeen en een deeltjes antigeen) en ingespoten via drie routes (intraperitoniaal, subcutaan en op de dorsale zijde van de achterpoot. De evaluatie is gebaseerd op klinische oobservaties, gedragsstudies, pathologische bevindingen en immunostimulatie. Lesies zijn het ernstigst na inspuiten van antigeen in Freund's adjuvant of Quil A, gering tot matig in combinatie met Specol en minimaal na Lactobacillus, RV-iscoms of fysiologisch zout. Ondanks de ernstige pathologische veranderingen, konden geen signalen van blijvende pijn of ongerief bij de dieren worden aangetoond aan de hand van klinische bevindingen en gedragsstudies. De immuunresponsen zijn zeer laag na inspuiten van antigeen in combinatie met fysiologisch zout of Lactobacillus. T-cel activatie en hoge antilichaam responsen zijn gevonden na inspuiten van antigeen/RV-iscoms conjugaten en na inspuiten van antigeen/Freund's adjuvant emulsies. T-cel activatie is aangetoond na inspuiten van antigeen/Specol emulsies en antilichaam responsen zijn hoog behalve na inspuiten van Specol in combinatie met autoantigeen. de gevonden resultaten suggereren dat Specol een mogelijk alternatief is voor Freund's compleet adjuvant voor de inductie van een immuunrespons tegen zwak immunogene antigenen behalve tegen autoantigenen, hiervoor lijkt RV-iscom een geschikt alternatief adjuvant.

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In anaphylactic reactions IgE-mediated effects on the pulmonary and cardiovascular system play a pivotal role. The sequence of amino acids of the chains of the IgE-antibody is known and decapeptides can be synthesized resembling that part of the chains which binds to the mast cells resulting in the release of histamine and leucotrienes. In theory these synthetic decapeptides can be used as the first step for the vaccine development. Another approach would be the construction of peptides with binding capacity but no stimulating activity resulting in the blocking of IgE-binding sites on the target cells. In this feasibility study pilot experiments were performed on the isolated heart of the (actively TNP-ovalbumin sensitized) rat with coronary flow as parameter. Although the pilot experiments indicated an approach leading to either effective peptide antagonists or vaccines it was foreseen that reaching this goal the demand on research capacity would be substantial. In defining priorities the final conclusion was reasched to end these experiments.

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In this study reduced food intake, food efficiency and growth were noticed at dose levels of 100 mg EAT/kg diet and higher. Slight changes in the red blood parameters were seen in the 100 and 500 mg EAT/kg diet dose groups. Urea concentration was increased in the females of the highest dose group. The urinalyses showed an increased urine production of male of the 100 and 500 mg/kg diet. Endocrinological analyses revealed decreased T4 (500 mg/kg diet) and TSH (100 and 500 mg EAT/kg diet, only in males). Macroscopical examination revealed gaseous distension of the duodenum, pale thyroids, red tail tips and enlarged iliacal lymph nodes. In the females of 20, 100 and 500 mg EAT/kg diet relative heart, brain and liver weights were higher than the control. The relative ovaries weights were increased at the 100 and 500 mg EAT/kg diet levels. In males (100 and 500 mg EAT/kg diet) relative heart and liver weights were elevated. A slight increase in regenerative and degenerative changes in the kidneys, strong activation of the iliacal lymph nodes was seen in the highest dose group. In the tail of the animal of the 500 mg EAT/kg diet group degenerative changes in the longitudinal skeletal muscle, sometimes accompanied by slight fibrosis was observed.

The department BES (Biological Evaluation of Drug Effects) carries out animal studies for RIVM laboratories, whose commitment to GLP makes it necessary for BES also to comply with GLP. Compliance is assured by means of a quality system laid down in a quality handbook quaranteeing the consistent quality of the department products. The elements of the BES quality system are documented in this report under the following headings: - quality targets and management organisation - survey of areas of competence and of stoff qualifications - equipment inventory - goods and services available - standard operating procedures. Furthermore a general servey is presented of those co-operative arrangements necessary for optimal departmental organization.

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Dit rapport is een aanvulling op het RIVM-rapport (nr. 749251001) van Leenhouts en Chadwick en is bedoeld als achtergrond-document voor het twee-mutatie-carcinogenese model en voor zijn implementatie. Het twee-mutatie-carcinogenese model is gebaseerd op een gemodificeerd twee-stappen model van Moolgavkar en Knudson in combinatie met een moleculair model dat cellulaire stralingseffecten beschrijft. In de eerste hoofdstukken van dit rapport worden het model en zijn implementatie beschreven. Het door Leenhouts en Chadwick beschreven twee-mutatie-carcinogenese model is een beetje gemodificeerd en de wiskundige onderbouwing en beperkingen van het model worden besproken. Verder zijn er optimalisatie-procedures aan de implementatie toegevoegd waarmee de modelparameters bepaald kunnen worden om de modelresultaten aan te passen aan epidemiologische of experimentele gegevens. Aan het eind van dit rapport wordt het model geillustreerd door het model toe te passen op het vormen van osteosarcoma's in honden (beagles) die geinjecteerd zijn met Ra-226. Het model kan zowel de leeftijds- als dosisafhankelijkheid van de osteosarcoma-genese goed reproduceren. In tegenstelling tot de aanname dat de dosis-respons relatie voor alfa-straling lineair is, voorspelt het model een niet-lineaire dosis-respons relatie voor de osteosarcoma-genese in beagles die geinjecteerd zijn met verschillende hoeveelheden Ra-226. Het model gaat er hierbij wel vanuit dat op cellulair niveau het aantal mutaties lineair gerelateerd is aan de door alfa-straling toegediende dosis. Verder blijkt uit het model dat de spontane incidentie van osteosarcoma's in beagles een grote invloed heeft op de dosis-respons relatie en dat een verandering in het dosisprofiel ook direct tot een andere dosis-respons relatie kan leiden. Deze invloeden worden momenteel niet meegenomen in de bepaling van stralingsrisico's.

Vier typen adjuvantia zijn geevalueerd als alternatief voor het gebruik van Freund's compleet adjuvant in muizen. De volgende adjuvantia zijn geevalueerd: een water-in-olie emulsie (Specol), een micro-organisme (Lactobacillus), een immuunstimulerend complex met ingebouwd glycoproteine van rabies virus (RV-iscoms) en een saponine. Quil A. De adjuvantia en controle groepen (fysiologisch zout) zijn gecombineerd met drie zwak immunogene antigenen (een synthetisch antigeen, een autoantigeen en een deeltjes antigeen) en ingespoten via drie routes (intraperitoniaal, subcutaan en op de dorsale zijde van de achterpoot. De evaluatie is gebaseerd op klinische oobservaties, gedragsstudies, pathologische bevindingen en immunostimulatie. Lesies zijn het ernstigst na inspuiten van antigeen in Freund's adjuvant of Quil A, gering tot matig in combinatie met Specol en minimaal na Lactobacillus, RV-iscoms of fysiologisch zout. Ondanks de ernstige pathologische veranderingen, konden geen signalen van blijvende pijn of ongerief bij de dieren worden aangetoond aan de hand van klinische bevindingen en gedragsstudies. De immuunresponsen zijn zeer laag na inspuiten van antigeen in combinatie met fysiologisch zout of Lactobacillus. T-cel activatie en hoge antilichaam responsen zijn gevonden na inspuiten van antigeen/RV-iscoms conjugaten en na inspuiten van antigeen/Freund's adjuvant emulsies. T-cel activatie is aangetoond na inspuiten van antigeen/Specol emulsies en antilichaam responsen zijn hoog behalve na inspuiten van Specol in combinatie met autoantigeen. de gevonden resultaten suggereren dat Specol een mogelijk alternatief is voor Freund's compleet adjuvant voor de inductie van een immuunrespons tegen zwak immunogene antigenen behalve tegen autoantigenen, hiervoor lijkt RV-iscom een geschikt alternatief adjuvant.

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In anaphylactic reactions IgE-mediated effects on the pulmonary and cardiovascular system play a pivotal role. The sequence of amino acids of the chains of the IgE-antibody is known and decapeptides can be synthesized resembling that part of the chains which binds to the mast cells resulting in the release of histamine and leucotrienes. In theory these synthetic decapeptides can be used as the first step for the vaccine development. Another approach would be the construction of peptides with binding capacity but no stimulating activity resulting in the blocking of IgE-binding sites on the target cells. In this feasibility study pilot experiments were performed on the isolated heart of the (actively TNP-ovalbumin sensitized) rat with coronary flow as parameter. Although the pilot experiments indicated an approach leading to either effective peptide antagonists or vaccines it was foreseen that reaching this goal the demand on research capacity would be substantial. In defining priorities the final conclusion was reasched to end these experiments.

Dit rapport is een aanvulling op het RIVM-rapport (nr. 749251001) van Leenhouts en Chadwick en is bedoeld als achtergrond-document voor het twee-mutatie-carcinogenese model en voor zijn implementatie. Het twee-mutatie-carcinogenese model is gebaseerd op een gemodificeerd twee-stappen model van Moolgavkar en Knudson in combinatie met een moleculair model dat cellulaire stralingseffecten beschrijft. In de eerste hoofdstukken van dit rapport worden het model en zijn implementatie beschreven. Het door Leenhouts en Chadwick beschreven twee-mutatie-carcinogenese model is een beetje gemodificeerd en de wiskundige onderbouwing en beperkingen van het model worden besproken. Verder zijn er optimalisatie-procedures aan de implementatie toegevoegd waarmee de modelparameters bepaald kunnen worden om de modelresultaten aan te passen aan epidemiologische of experimentele gegevens. Aan het eind van dit rapport wordt het model geillustreerd door het model toe te passen op het vormen van osteosarcoma's in honden (beagles) die geinjecteerd zijn met Ra-226. Het model kan zowel de leeftijds- als dosisafhankelijkheid van de osteosarcoma-genese goed reproduceren. In tegenstelling tot de aanname dat de dosis-respons relatie voor alfa-straling lineair is, voorspelt het model een niet-lineaire dosis-respons relatie voor de osteosarcoma-genese in beagles die geinjecteerd zijn met verschillende hoeveelheden Ra-226. Het model gaat er hierbij wel vanuit dat op cellulair niveau het aantal mutaties lineair gerelateerd is aan de door alfa-straling toegediende dosis. Verder blijkt uit het model dat de spontane incidentie van osteosarcoma's in beagles een grote invloed heeft op de dosis-respons relatie en dat een verandering in het dosisprofiel ook direct tot een andere dosis-respons relatie kan leiden. Deze invloeden worden momenteel niet meegenomen in de bepaling van stralingsrisico's.

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The department BES (Biological Evaluation of Drug Effects) carries out animal studies for RIVM laboratories, whose commitment to GLP makes it necessary for BES also to comply with GLP. Compliance is assured by means of a quality system laid down in a quality handbook quaranteeing the consistent quality of the department products. The elements of the BES quality system are documented in this report under the following headings: - quality targets and management organisation - survey of areas of competence and of stoff qualifications - equipment inventory - goods and services available - standard operating procedures. Furthermore a general servey is presented of those co-operative arrangements necessary for optimal departmental organization.

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A subchronic toxicity experiment with propyl gallate in SPF-derived Wistar RIVM:TOX rats was performed. Groups of 10 female and 10 male rats were fed a semisynthetic diet containing 0, 490, 1910 and 7455 mg propyl gallate /kg feed. Body weight gain was recorded weekly and food-intake twice weekly. Other parameters comprised haematology, biochemical determinations in urine, serum and liver and complete histopathological examinations. Adverse effects of propyl gallate observed in the high dose group were effects on the haemopoeitic system reflected in the haematological parameters and the morphological changes in the spleen. The other effects observed comprised decreased incidence of the nephrocalcinosis in female rats, the increased activity of EROD in the high dose group and increased activity of the conjugating enzymes; glucorynyl-transferase and glutathion-s-transferase, in the mid and high dose group of propyl gallate. The effects on the nephrocalcinosis and on the conjugating enzymes may be considered as not adverse. The no observed adverse effect level (NOAEL) is 1910 mg propyl gallate /kg feed corresponding with 135 mg propyl gallate /kg body weight.

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Eight groups of six male RIV:tox rats received oral and intravenous doses of benzo(a)pyrene (BaP) in a parallel study plan. Intravenously and orally four dose levels were administered. BaF was dissolved in soybean oil for oral administration, and in glycofurol for intravenous administration. The doses were such, that the absolute oral bioavailability could be calculated for two dose levels. The plasma pharmacokinetics of BaP after intravenous administration were described by a two-compartment model. The corresponding kinetic parameters were calculated. The elimination half-life of BaP was approx. 30-60 min. in the period 0-12 h after administration. Therefore, unchanged BaP will not accumulate after repeated administration, when it is administered once daily. The AUC values (area under the plasma concentration-time curve) increased more than proportionally with the oral or intravenous dose. The deviation from linearity was small for the doses used in this study. After oral administration multiple plasma concentration maxima were observed, which may be explained by an interaction of the oil solution with the bile. The absolute oral bioavailability of unchanged BaP was approx. 13% for a dose of 1 mg/kg and approx. 4% for a dose of 5 mg/kg. This does not mean, that the absorption of BaP decreased with increasing doses, because the non-linear increase of the AUC values with the dose may be explained by saturation of metabolism. The kinetic parameters and the oral bioavailability were in the same range as found in the literature for other rat species.

Eight groups of six male RIV:tox rats received oral and intravenous doses of benzo(a)pyrene (BaP) in a parallel study plan. Intravenously and orally four dose levels were administered. BaF was dissolved in soybean oil for oral administration, and in glycofurol for intravenous administration. The doses were such, that the absolute oral bioavailability could be calculated for two dose levels. The plasma pharmacokinetics of BaP after intravenous administration were described by a two-compartment model. The corresponding kinetic parameters were calculated. The elimination half-life of BaP was approx. 30-60 min. in the period 0-12 h after administration. Therefore, unchanged BaP will not accumulate after repeated administration, when it is administered once daily. The AUC values (area under the plasma concentration-time curve) increased more than proportionally with the oral or intravenous dose. The deviation from linearity was small for the doses used in this study. After oral administration multiple plasma concentration maxima were observed, which may be explained by an interaction of the oil solution with the bile. The absolute oral bioavailability of unchanged BaP was approx. 13% for a dose of 1 mg/kg and approx. 4% for a dose of 5 mg/kg. This does not mean, that the absorption of BaP decreased with increasing doses, because the non-linear increase of the AUC values with the dose may be explained by saturation of metabolism. The kinetic parameters and the oral bioavailability were in the same range as found in the literature for other rat species.