EuroFIR eBASIS: application for health claims submissions and evaluations.

BACKGROUND: The European Food Information Resource (EuroFIR) network has established the eBASIS (Bioactive Substances in Food Information System) online food composition and biological effects database for plant-derived bioactive compounds (phytochemicals). On the basis of submitted evidence, the European Food Safety Authority (EFSA) expert panel on Dietetic Products, Nutrition and Allergies assesses whether claims made under articles 13.1, 13.5 or 14 of the Regulation (EC) 1924/2006, which governs the use of nutrition and health claims on foods, are scientifically justified. This report evaluates the eBASIS biological effects database in the preparation and evaluation of health claims dossiers. METHODS: The eBASIS biological effects database is a compilation of expert-evaluated data extracted from the literature, prioritizing human intervention studies to investigate health effects of phytochemicals. Currently included are >750 records from 445 studies providing data on 56 validated biomarkers, mainly relating to cardio-metabolic and bone health outcomes. The data cover 144 bioactive compounds from 17 compound classes. Using the EFSA Register of Questions and the database of general function health claims, we identified claims relating to phytochemicals made under articles 13.1, 13.5 and 14 and compared them with the eBASIS database to identify overlap between them. RESULTS: The EFSA online health claims database contains 4240 submissions under article 13.1, of which 2157 pertain to plants or plant-based bioactive compounds; 496 of these relate to plants or bioactive compounds included in the eBASIS biological effects database. Out of the 18 current 13.5 'new function' claims on EFSA's register of questions, 7 are for plants or plant-based bioactive compounds, of which 6 are included in eBASIS. Of the 222 defined article 14 claims, 21 pertain to plants or plant-based bioactive compounds, of which 19 are in eBASIS. CONCLUSIONS: There is extensive overlap between eBASIS and the submitted health claims that relate to plant-based bioactive compounds. EuroFIR eBASIS is a useful tool for regulators to independently check completeness of health claims applications relating to phytochemicals and is a potentially valuable resource to assist claimants in the compilation of dossiers on functional foods and health claims.

Combined toxic effects of mycotoxins.

It is known for many years that several food items, derived from plants infected by fungi in the field during growing of the plant or during harvest and storage of the food item, can contain concomitantly different mycotoxins. As these combined mycotoxins occur simultaneously in the food item, consumption of the food will lead to a combined intake depending on the absorption rates of the different mycotoxins. Therefore, the question is justified whether such a combined intake of mycotoxins would lead to a possible higher risk for adverse health effects than the intake of one of these mycotoxins alone. It will be dealt with on the basis of some practical cases of such combined intake of mycotoxins of which research data are available. This is the case for citrinin and ochratoxin A, but as the workshop focuses on trichotecenes and so this paper concentrates on these. When the mycotoxins are of similar structure and of the same species, or of the same families, it is likely to expect that the mode of action of the mycotoxins and or the toxicity profiles will be quite similar. This indicates that such related mycotoxins are likely to exert only additive effects, which is important to know. In terms of risk assessment, these mycotoxins could be dealt with by establishing a group daily tolerable intake (TDI) or a provisional tolerable weekly intake (PTWI). In terms of risk assessment those mycotoxins which interact in synergistic manner are of more concern. It is concluded that, at present tools are not fully developed to establish the type of interaction or whether there is any interaction at all.

Age-related differences in the toxicity of ochratoxin A in female rats.

Ochratoxin A (OTA) is a mycotoxin found in food and feedstuffs of plant and animal origin. OTA exposure is related to nephropathy in humans. Age-related differences, especially in nephro- and immunotoxicity of OTA, were investigated in young adult (aged 12 weeks) and old (aged 27-30 months) female SPF Wag rats, treated by gavage with 0, 0.07, 0.34 or 1.68 mg OTA/kg body weight for 4 weeks. In both age groups, survival was significantly decreased in the highest dose group. Clinical condition, body weight, clinical chemistry parameters (ALAT, ASAT, creatinin and urea) and target organs (as identified by weight and pathology - kidney, liver, adrenals, forestomach and brain) were affected by age and dose, but often more severely in old than in young rats. OTA induced primarily nephropathy. Old rats were more sensitive to induction of tubular karyomegaly and vacuolation/necrosis. In young rats, OTA induced a dose-related thickening of the basement membrane and reduction in splenic T-cell fraction. Decreased IgG levels were seen at 0.34 mg/kg OTA (young and old rats) and 1.68 mg/kg OTA (young rats). Vacuolation of the white brain matter (cerebellar medulla and ventral parts of the brain stem) was significantly increased in young rats at 0.34 and 1.68 mg/kg OTA and in old rats at 0.07 and 0.34 mg/kg OTA. It was concluded that: (1) the profiles of OTA toxicity for both age groups are similar, with the kidney and possibly the brain being primary target organs; (2) based on clinical and pathological data old rats are more sensitive to OTA than young rats; and (3) the immune system is probably not the primary target of OTA toxicity.

Subacute toxicity of alpha-ergocryptine in Sprague-Dawley rats. 1: general toxicological effects.

The dietary subacute toxicity of the ergot alkaloid alpha-ergocryptine was studied in Sprague-Dawley rats. Rats were fed 0, 4, 20, 100 or 500 mg ergocryptine/kg diet for 28-32 days (equal to 0, 0.36, 1.7, 8.9 and 60 mg ergocryptine/kg body weight/day for females and 0, 0.34, 1.4, 6.6 and 44 mg ergocryptine/kg body weight/day for males). The present study describes the general toxicological effects; the effects on metabolic and hormonal parameters will be reported separately. Body weight, body weight gain, food intake and food efficiency were all decreased with a U-shaped dose-response curve, as in both sexes the ranking severity of effects was in the order 100-20-500 and 4 mg/kg diet. Other changes with a U-shaped dose-response relationship included: hematological parameters (decreased MCV and MCH), serum enzyme activities (slightly increased/decreased ALAT, ASAT, GGT), increased serum urea concentrations, decreased glomular filtration (creatinine and urea clearances), decreased absolute organ weights, increased and decreased relative organ weights, atrophy of thymus and in females atrophy of ovary and uterus with in the mid-dose groups no detectable morphological features of an oestric cycle in the uterus. Other parameters, including increased relative liver, heart and ovarian weights and necrosis of the tail, were influenced in a dose-related manner or only in the high dose group. The U-shaped changes for the parameters mentioned above might be caused by the U-shaped dose-response relationship for food intake, which may be explained by the dopaminergic properties of alpha-ergocryptine. It is concluded that in rats fed ergocryptine for 28 days the dose-effect curve is rather steep and that the NOAEL is 4 mg/kg diet.

Subacute toxicity of alpha-ergocryptine in Sprague-Dawley rats. 2: metabolic and hormonal changes.

The present study describes the metabolic changes observed in a dietary subacute toxicity experiment with the ergot alkaloid alpha-ergocryptine in Sprague-Dawley rats. The observed effects on metabolic and hormonal parameters were described separately from the general toxicological effects, in view of the important role of dopamine agonists in metabolism (e.g. ergot alkaloids in fescue toxicosis). The rats were fed 0, 4, 20, 100 or 500 mg ergocryptine/kg diet for 28-32 days (equal to 0, 0.36, 1.7, 8.9 and 60 mg ergocryptine/kg body weight/day for females and 0, 0.34, 1.4, 6.6 and 44 mg ergocryptine/kg body weight/day for males). Total cholesterol and high-density lipoprotein (HDL)-cholesterol were decreased dose dependently in females but the ratio HDL-cholesterol/total cholesterol was only decreased at 20 mg/kg body weight. Triglycerides and glucose concentrations were decreased in the highest dose groups of both sexes. Serum urea concentrations were increased in the 20, 100 and 500 mg/kg dose groups. Insulin, glucagon and liver glycogen were increased in the highest dose group at the end of the study, when the animals were allowed to eat prior to blood sampling and necropsy. Prolactin, T4 and FT4 were decreased in the 20, 100 and 500 mg/kg dose groups of both sexes. Follicle-stimulating hormone (FSH) was decreased in the 20, 100 and 500 mg/kg female dose groups and luteinizing hormone (LH) was increased in the 20, 100 and 500 mg/kg male dose groups. It is postulated that the observed effects on food intake, metabolism (lipid and carbohydrate) and hormonal parameters are due to an interaction of ergocryptine with central dopaminergic activities, which comprise a major functional component of a central regulatory system for metabolism.

Precision of estimates of an ADI (or TDI or PTWI).

Factors influencing the precision of an acceptable daily intake (ADI) are discussed in this paper. As the same principles apply to tolerable daily intake (TDI) or provisional tolerable weekly intake (PTWI), although not specifically mentioned, this paper also refers to TDI and PTWI. The allocation of an ADI is in principle based on the most critical (many times the lowest) no-observed (adverse)-effect level [NO(A)EL] established in toxicological studies in experimental animals or in humans by applying a uncertainty factor for extrapolation from animals or humans to the general human population (and for the extrapolation from high to low intake levels). As the ADI predicts a virtual safe intake level for a life span exposure, to establish a NO(A)EL in general the toxicological database should include long-term studies, otherwise only a provisional ADI will be allocated for which a higher uncertainty factor is applied. The validity of an ADI greatly depends on the precision of the toxicological studies considered for the safety of a food additive or contaminant. The precision of the ADI is also inversely related to the uncertainty factors applied, although these uncertainty factors are not totally independent of the completeness and precision of the toxicological data from which a NO(A)EL is derived. This paper focuses on the precision of the toxicological data and the established NO(A)EL. Human data on the toxicity of a chemical which are preferred for the safety evaluation or hazard assessment are frequently not available or incomplete with respect to a quantitative dose-response assessment. Epidemiological studies will have inherent difficulties for hazard assessment such as possible confounders, restricted number of toxicological end points which can be studied, and limited quantitative data on oral exposure levels. Case report studies include the same limitations but in addition the exposure data are usually very imprecise due to reconstruction of the possible dose level(s). Case reports of intoxication are mainly restricted to acute and at best subacute effects. Controlled human exposure studies (human volunteer studies) are restricted in their experimental design such as the level of the dose and the toxicological end points due to medical ethical reasons. Therefore, quite rarely a safety evaluation of a food chemical will be solely based on human data. In the practice of hazard assessment of chemicals in foods the experimental animal studies will be totally or partly the basis for establishing an ADI. In these toxicological studies in animals there are many experimental variables which can affect the precision of an ADI, such as (1) duration of the experiment, dose ranges, identity, and purity of the substance; (2) the parameters and toxicological end points studied; (3) the species and strain used; (4) the gut microflora of the test animals; (5) dietary composition; (6) statistics performed; and (7) knowledge about the kinetic behavior and metabolism (e.g., elimination half-life and bioavailability of the chemical and its main metabolites) of the chemical considered. How these factors can influence the precision of a NO(A)EL, respectively the ADI, is illustrated by several examples. In relation to the question of incidental excursions of an ADI, it can be concluded that due to the variation in precision of experiments slight incidental excursions would not lead to an increased risk. However, to answer in general the question of how often and/or how much the total intake of a chemical in food may exceed the ADI is not possible. This should be considered case by case. To answer such a question, the precision for those studies representative for incidental excursions should be considered. Other factors which should be considered are (1) type of effect on which the ADI was based, (2) mechanism of toxicity, (3) toxicokinetics and metabolism, and (4) difference in NO(A)ELs from short-term toxicity studies with the NO(A)EL on

Comparison of tobramycin nephrotoxicity in young adult and aged female rats.

A two week toxicity study was performed in rats to study the possible age-dependent toxicity of tobramycin, an aminoglycoside antibiotic with well known ototoxic and nephrotoxic properties in animals and man. Young adult female Wag/Rij rats aged 12 weeks (n = 10) and old female rats aged 23 to 26 months (n = 14) were treated subcutaneously with 0, 10, 40 or 160 mg tobramycin sulphate/kg/day. Clinical chemistry and urinalysis revealed significant changes in renal function in young adult rats mainly at 160 mg/kg, whereas in old rats significant changes were seen at 10, 40 and 160 mg/kg. Excretion of N-acetyl-beta-glucosaminidase, indicative for tubular dysfunction, was statistically significantly increased only in old animals at 160 mg/kg. Histopathology: At 40 mg/kg, tubular necrosis was increased in old animals and hyaline droplet formation in both age groups. At 160 mg/kg these lesions were increased in both age groups. For tubulonephrosis, interstitial nephritis and tubular regeneration, age-related differences were predominantly reflected in severity, for example, at 40 mg/kg, tubular regeneration in young animals was "moderate" in 7/10 and "marked" in 2/10, while in old animals the scores were 3/14 and 11/14, respectively. Secondary treatment-related lesions (in heart and adrenals) were also more increased in old animals. Chemistry and histopathology revealed the increased sensitivity to the toxic effects of tobramycin in old rats, which is important for the discussion of the most appropriate dosing regimen for aminoglycoside in humans. The once-daily dosing regimen for tobramycin should not be recommended for elderly, because high peak concentrations should be avoided to minimise nephrotoxicity.

Food plant toxicants and safety Risk assessment and regulation of inherent toxicants in plant foods.

The ADI as a tool for risk management and regulation of food additives and pesticide residues is not readily applicable to inherent food plant toxicants: The margin between actual intake and potentially toxic levels is often small; application of the default uncertainty factors used to derive ADI values, particularly when extrapolating from animal data, would prohibit the utilisation of the food, which may have an overall beneficial health effect. Levels of inherent toxicants are difficult to control; their complete removal is not always wanted, due to their function for the plant or for human health. The health impact of the inherent toxicant is often modified by factors in the food, e.g. the bioavailability from the matrix and interaction with other inherent constituents. Risk-benefit analysis should be made for different consumption scenarios, without the use of uncertainty factors. Crucial in this approach is analysis of the toxicity of the whole foodstuff. The relationship between the whole foodstuff and the pure toxicant is expressed in the `product correction factor' (PCF). Investigations in humans are essential so that biomarkers of exposure and for effect can be used to analyse the difference between animals and humans and between the food and the pure toxicant. A grid of the variables characterising toxicity is proposed, showing their inter-relationships. A flow diagram for risk estimate is provided, using both toxicological and epidemiological studies.

Healthy volunteer studies in toxicology.

In the future there will be an increasing need for quantitative human risk assessment in order to develop soundly-based risk level regulations. Human volunteer studies can contribute to gain essential data needed for this risk assessment. Volunteer studies are especially relevant to study the biokinetics and metabolism of a compound. Comparison of these data with those of laboratory animals can increase the accuracy in extrapolation study results from animals to man. Furthermore, the results of volunteer studies can be used to fill in the gaps of knowledge which cannot be solved with in vitro or animal studies in order to develop adequate physiologically-based biokinetic or biodynamic models for human risk assessment.

Subacute toxicity of ergometrine maleate in rats.

Ergot alkaloids, produced by the fungus Claviceps purpurea, are found in small amounts in foodstuffs. The human disease ergotism, caused by high intake of ergot alkaloids, is well known; however, little is known about the toxicity of these compounds. The subacute toxicity of an ergot alkaloid, ergometrine maleate, was therefore studied. Sprague-Dawley rats were treated with 0, 2, 10, 50 and 250 mg ergometrine maleate/kg diet for 4 wk. Plasma glucose levels were decreased in females at 50 and 250 mg/kg. Thyroxin levels were decreased at 50 (males only) and 250 mg/kg. At the high dose level, organ weights of heart, liver, ovaries and kidneys were increased. In male rats a slight dose-related increase in the incidence of enlarged mediastinal lymph nodes and, to some extent, of enlarged parathymal lymph nodes, was seen. Histopathological examination revealed evidence of increased glycogen storage in the liver of animals treated with 250 mg/kg. The no-observed-effect level in this study was 10 mg/kg.

Cadmium and atherosclerosis in the rabbit: reduced atherogenesis by superseding of iron?

This study evaluated the effect of dietary cadmium (Cd) on atherosclerosis in the rabbit. Cholesterol was added to the diet to initiate and/or accelerate atherogenesis. Cd was added to the diet at two dose levels. Uptake of Cd was 55 micro gram/kg body weight (BW)/day at the low dose level and 1350 micrograms/ kg BW/day at the high dose level. Five groups of rabbits were fed five different diets for 9 months: (1) basal diet without additional constituents; (2) background diet, which was basal diet to which cholesterol had been added; (3) the low-dose level Cd diet, which was background diet to which 2 mg Cd/kg had been added; (4) high-dose level Cd diet, which was background diet to which 50 mg Cd/kg had been added; and (5) basal diet to which 50 mg Cd/kg had been added. Dietary cholesterol increased blood total leucocyte count, serum and liver total cholesterol concentrations, serum total bilirubin concentration, low-density lipoprotein vitamin E concentration and induction of atherosclerotic plaques in the aorta and coronary arteries. Cd in the diet increased liver and kidney Cd concentrations in a dose-dependent way, decreased prothrombin time and temporarily increased urea and creatinine clearances. Slight kidney damage was induced by Cd only in animals fed the high-dose level Cd diet (with or without cholesterol). Dietary Cd partly counteracted the dietary cholesterol-induced increases of serum and liver total cholesterol concentrations, and tended to reduce plaque formation in the aorta. Dietary Cd in rabbits fed cholesterol-containing diets influenced cholesterol metabolism and tended to decrease atherosclerosis in a dose-related fashion. This is in contrast with limited epidemiological human data. Dietary Cd also decreased serum ferritin concentration and increased serum transferrin concentration. Free iron concentration is associated with myocardial infarction in man and augments the development of atherosclerosis in rabbits. It is concluded that the observed reduction in atherogenesis is related to dietary Cd-induced changes in cholesterol metabolism, increased rheology of blood and/or, most likely, reduced free iron concentration.

Ototoxicity of tobramycin in young adult and old rats.

To study the possible age-dependent ototoxic effects of tobramycin a subacute toxicity study was performed. To young adult (3 months) and old (27-30 months) female Wag-Rij rats 0, 10, 40, and 160 mg tobramycin sulfate/kg was administered subcutaneously in two doses a day. After 14 days all animals were autopsied. The cochlea was fixed by perfusion through the opened oval window and prepared for scanning electron microscopy. The rat cochlea consists of two turns. In all animals of the young control group the row of inner hair cells (IHC) as well as the three rows of outer hair cells (OHC) of the organ of Corti were fully intact. IHC as well as OHC are provided with three rows of stereocilia, from inside to outside with increasing length. The stereocilia of OHC are arranged in a characteristic W-configuration. In the young low dose group effects were focally seen in only 1 of 10 rats, consisting of shortened, disoriented or partly disappeared stereocilia of IHC and OHC in the basal turn. In the young mid dose group 4 of 10 animals had focally shortened, disoriented, and less stereocilia in the first row of OHC in the basal turn, and once of IHC and OHC in the apical turn. Seven of 8 animals of the young high dose group showed effects consisting of focal loss or shortening of stereocilia of IHC and OHC of the apical turn (3x), of OHC in the basal turn (3x), and once of stereocilia of IHC only. In the old control group the stereocilia of IHC were fully intact. However, many OHC, independent of row or turn, had no stereocilia at all. The percentages of OHC without stereocilia in the three rows of the apical and basal turns were 44-10-50 and 40-20-50, respectively. In the old low, mid, and high dose group the percentages of OHC without stereocilia were nearly identical to those of the old control group. In the old low and high dose group a reduced number of stereocilia per IHC occurred in half of the number of animals, while in the mid dose group the IHC were fully intact. In young adult animals the number of mildly affected cochleae increased as well as the extent of the lesion increased with increasing dose of tobramycin. The lesions of IHC and OHC, consisting of a decrease in number or shortening of stereocilia, were restricted mainly to (the last part of) the basal turn. As old control rats showed already a large number of OHC without stereocilia, the possible ototoxic effects of tobramycin were not detected against this "background" of stereociliary loss, consistent with aging. The damage in old rats was certainly not greater than in young adult rats. In conclusion, old rats were not more sensitive to the possible ototoxic effects of tobramycin than young adult rats.

Toxicological data needed for safety evaluation and regulation on inherent plant toxins.

Although the awareness that natural compounds in foodstuffs can have implications for human health is growing, there is a lack of data on the toxicology and occurrence. To enable the safety evaluation of inherent plant toxins adequate toxicological data are necessary. Of most inherent plant toxins at best only limited toxicological data are available, which makes it impossible to perform an accurate safety evaluation. This limited knowledge of inherent plant toxins permits the mystical claim of safety on the basis of history of food use, and thus the development of specific food safety regulation has been postponed. To further develop flexible and adequate regulation, for at least some inherent plant toxins, an adequate set of toxicological data should be available in order to verify and validate the initial regulation. Several problems (e.g., availability of the plant toxin or accuracy of historical human toxicity data) which arise when gathering the necessary data and the development of regulations are discussed and elaborated with a few examples such as solanum alkaloids and glucosinolates. As long as nobody is held responsible to study safety aspects of inherent plant toxins, the lack of data will persist. Therefore a certain level of regulation should be recommended to provide the data needed. Such safety research projects integrated with new agricultural developments should be endorsed by the European Union and other international bodies.

Nitrate, nitrite and N-nitroso compounds.

A risk assessment has been made on nitrate, nitrite and N-nitroso compounds encountered in the human diet. Vegetables constitute a major source of nitrate providing over 85% of the average daily human dietary intake. Nitrite and N-nitroso compounds present in the diet contribute relatively small amounts to the body burden and the major source of these biologically reactive compounds is derived from the bacterial and mammalian metabolism of ingested nitrate. Additionally, endogenous synthesis provides an important source contributing to the body burden of nitrate. Data from animal toxicological studies, human effects and epidemiological surveys have been reviewed and evaluated. It is concluded that there is no firm scientific evidence at present to recommend drastic reductions beyond the average levels of nitrate encountered in vegetables grown in keeping with good agricultural practice. Recommendations have also been made for further animal and human studies to be carried out to elucidate the potential risks to man from ingested nitrate.

Bioavailability and disposition of 3H-solanine in rat and hamster.

1. The toxicokinetics of [3H]-alpha-solanine after oral (p.o.) and intravenous (i.v.) administration in rat and hamster were studied, in order to decide which is the most appropriate model in risk assessment studies. The i.v. dose was 54 micrograms/kg; the oral dose was 170 micrograms/kg. 2. After i.v. administration, the toxicokinetics of total radioactivity in blood were comparable in rat and hamster. However, the clearance of total radioactivity from plasma was more effective in rat than in hamster. The half-lives of distribution and of the terminal phase of unchanged alpha-solanine were not different between rat and hamster, whereas the systemic and metabolic clearance were, respectively, about 1.6 and 2.7 times higher in rat than in hamster. The clearance of unchanged alpha-solanine is more effective than of total radioactivity. 3. After p.o. administration in rat and hamster, the mean bioavailability of total radioactivity is about 29 and 57%, respectively. The bioavailability of unchanged alpha-solanine is only 1.6 and 3.2%, respectively, when compared with i.v. administration. 4. T1/2el of alpha-solanine after p.o. administration was in rats a factor of four and in hamsters a factor of two shorter than after i.v. administration. A strong retention of radioactivity was seen in the hamsters after p.o. administration; only 40% of the dose was excreted within 7 days versus 90% in rat. 5. Based on these and toxicological data from literature, it was decided that the hamster is a more appropriate model in (sub)-chronic toxicity studies with alpha-solanine than the rat.

Is dietary ergotamine atherogenic in the rabbit?

The effect of dietary ergotamine on pre-lesional indicators of atherosclerosis was studied in rabbits. The experimental purified diets contained 0.08% (w/w) cholesterol and either 0, 40 or 200 mg ergotamine-tartrate/kg. After 6 wk, serum total cholesterol concentration and the ratio of serum total cholesterol: high-density lipoprotein-cholesterol were significantly increased by ergotamine in a dose-dependent manner. Dietary ergotamine raised the total level of glycosaminoglycans and the relative proportion of chondroitin sulphate in the abdominal aorta. It is suggested that dietary ergotamine is atherogenic in the rabbit.

Subacute toxicity of Zwittergent administered intramuscularly.

In order to assess the systemic toxicity of Zwittergent 3-14, a detergent used in the protein-detergent complex vaccines for its attractive immunogenic properties, a subacute toxicity study was performed. In this 4-week toxicity experiment five female and five male rats per group were injected intramuscularly with 0.25 ml of 0, 75, 750 and 7500 micrograms Zwittergent ml-1 sterile saline solution. Body weight and food intake were recorded weekly. At day 24 urine was collected for semiquantitative (pH, protein, ketone bodies, bilirubin and occult blood) and quantitative analyses (protein, creatinine and volume). At the end of the experiment blood was sampled for haematological [haemoglobin (Hb), packed cell volume (PCV), erythrocytes, leucocytes, reticulocytes and thrombocytes and differential white blood cell count] and biochemical analyses (alanine aminotransferase and aspartate aminotransferase. At necropsy brain, heart, liver, kidneys, spleen, thymus, adrenal glands, thyroid, pituitary gland, uterus, ovaries and testes were weighed. The underlined organs and the musculus quadriceps including the injection sites were examined histopathologically. Indications for systemic toxicity were noticed in the high-dose group and included occult blood in urine, elevated protein/creatinine ratio concomitantly with an increased urine volume and increased relative kidney weight indicating a slight disturbance of the kidney function. Some changes in haematological parameters (decreased PCV and increased numbers of thrombocytes eosinophils and monocytes) and a decreased glycogen content in the liver were recorded in the high dose group. These changes may be secondary to the extensive inflammatory reaction observed in the muscle of this high-dose group.(ABSTRACT TRUNCATED AT 250 WORDS)

Local reactions of Zwittergent-containing meningococcal vaccine after intramuscular injection in rats: comparison with the effect of diphtheria-pertussis-tetanus-polio vaccine.

A short-term toxicity study was performed to investigate local reactions and haematological changes after intramuscular (i.m.) injection of meningococcal vaccine with a concentration of 25 or 50 micrograms protein ml-1. In these meningococcal vaccines up to 75 micrograms Zwittergent ml-1 was used to form a protein-detergent complex with attractive immunogenic properties. The Zwittergent preparations were treated with NaOH in order to destroy any propane sultone residue and this was checked by HPLC. The effects were compared with those caused by a sterile phosphate-buffered saline solution and by diphtheria-pertussis-tetanus-polio (DPT-polio) vaccine. Groups of six male rats were injected i.m. with 0.25 ml of the test solution, on day 0 in the left, and on day 7 in the right, quadriceps muscle. On day 14 the animals were exsanguinated under ether anaesthesia and, after gross inspection, inguinal nodes were weighed. These lymph nodes and the left and right quadriceps muscles were studied microscopically. In addition, a haematological investigation was performed. A significant increase in the number of thrombocytes was seen in animals receiving the DPT-polio and meningococcal vaccines containing 25 micrograms protein ml-1, and an increase in the number of leucocytes, due to an increase of neutrophils and monocytes, was seen in animals receiving the DPT-polio and meningococcal vaccine containing 50 micrograms protein ml-1. In comparison with the DPT-polio vaccine (a product generally acceptable for use in man), the local inflammatory reaction in the muscles was less in rats injected with the meningococcal vaccines (especially in the 25 micrograms protein ml-1 group).(ABSTRACT TRUNCATED AT 250 WORDS)