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Pharmaceuticals and chemicals are subjected to regulatory safety testing accounting for approximately 25% of laboratory animal use in Europe. This testing meets various objections and has led to the development of a range of 3R models to Replace, Reduce or Refine the animal models. However, these models must overcome many barriers before being accepted for regulatory risk management purposes. This paper describes the barriers and drivers and options to optimize this acceptance process as identified by two expert panels, one on pharmaceuticals and one on chemicals. To untangle the complex acceptance process, the multilevel perspective on technology transitions is applied. This perspective defines influences at the micro-, meso- and macro level which need alignment to induce regulatory acceptance of a 3R model. This paper displays that there are many similar mechanisms within both sectors that prevent 3R models from becoming accepted for regulatory risk assessment and management. Shared barriers include the uncertainty about the value of the new 3R models (micro level), the lack of harmonization of regulatory requirements and acceptance criteria (meso level) and the high levels of risk aversion (macro level). In optimizing the process commitment, communication, cooperation and coordination are identified as critical drivers., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The Organisation for Economic Co-operation and Development has undertaken an international validation program for the rodent uterotrophic bioassay. This validation program comprised two major parts. The first part was the development of a detailed background review document compiling the existing data on the bioassay's history, the molecular and physiologic basis for the bioassay's mechanistic relevance to detect estrogen agonists and antagonists, a review of important bioassay protocol parameters, and a review of the data generated by in vitro assays, previous uterotrophic bioassays, and developmental and reproductive assays to assess and support the overall predictivity of the uterotrophic bioassay. The second part was an extensive multiyear effort managed by a validation management group to demonstrate the operating characteristics of four protocols. The effort was conducted in two phases. The phase 1 results with the reference agonist ethinyl estradiol (EE) and antagonist ZM 189,154 has been published previously. This Environmental Health Perspectives mini-monograph is devoted to the phase 2 work using five weak estrogen agonists, bisphenol A, genistein, methoxychlor, nonylphenol, and o,p -DDT, as well as the negative substance dibutylphthalate. These data show that all protocols successfully detected increases in uterine weights when a sufficient dose level of the weak agonists was administered, whether the substances were known or provided as coded doses to the laboratory. The data with both the reference EE and all five weak agonists are reproducible over time and under a variety of different experimental conditions (e.g., animal strain, diet, housing, bedding, vehicle, animal age). In conclusion, all protocols now have sufficient data to support their validity.

An essential aspect of the OECD is that it should not be considered a supranational organisation, but rather a center for discussion where governments express their points of view, share their experiences and search for common ground. This implies that decisions are made by consensus instead of majority. Once the Council, which is the highest authority of the OECD, adopts a formal Decision, such a decision is binding on all Member countries. The OECD Guidelines for the Testing of Chemicals, which are considered the leading international standard for safety testing, form an integrated part of such a binding Council Decision. An even more important part of that same Council Decision is that on Mutual Acceptance of Data, where it states that: 'Data generated in the testing of chemicals in an OECD Member country in accordance with OECD Test Guidelines and OECD Principles of Good Laboratory Practice (GLP) shall be accepted in other Member countries for purposes of assessment and other use relating to the protection of man and the environment.' In the various steps of the process of Test Guideline development, the National Co-ordinators of the Test Guideline Programme play an important role. The initiative to start the development of a particular guideline can be taken by the OECD Secretariat, by one or more Member countries or, most importantly, by the scientific community itself. Proposals, received by the Secretariat are discussed at the yearly Meeting of the National Co-ordinators. During these meetings, priorities for future activities are set and the approach that should be followed in dealing with the selected activities is discussed. Quite often, so-called Detailed Review Papers (DRP's) form the basis of a new or updated Guideline. These DRP's, which are either prepared by a Member country or by a consultant appointed by the Secretariat, describe the current 'state of the art' in scientific progress and technical possibilities of a well-defined area of research. After completion, either an expert meeting or a commenting round will be organised. All Member countries will have sufficient possibilities to express their views. When the DRP is acceptable to the experts of all Member countries, the next step is to actually develop a Test Guideline. Similar to the procedure followed for the DRP, the Test Guideline proposal will be circulated for comment to all Member countries and should reach the desks of relevant experts, nominated by their National Co-ordinator. Frequently, in addition to the commenting rounds, Test Guideline proposals are discussed in special expert meetings. Once the experts reach consensus on a particular Test Guideline, the proposal is put forward to the Meeting of the National Co-ordinators for approval. Since each guideline will form an integrated part of the earlier mentioned Council Decision, each new guideline also needs formal adoption by the Council before it becomes effective.

A personal view is presented on progress made during the last 25 years in applying the Three Rs (reduction, refinement, replacement) to animal testing in regulatory toxicology, with an emphasis on "good moments" (for example, international workshops on the principles and practical application of the validation process and on regulatory acceptance) and "not-so-good moments" (for example, the time taken to accept alternatives to the LD50 test and to accept in vitro tests for skin absorption as OECD Test Guidelines). The importance of dialogue and cooperation between international coordinating centres and scientific activities at the national level is stressed, as exemplified by the work of ECVAM during its first decade.

This paper is one of several prepared under the project "Food Safety In Europe: Risk Assessment of Chemicals in Food and Diet" (FOSIE), a European Commission Concerted Action Programme, organised by the International Life Sciences Institute, Europe (ILSI). The aim of the FOSIE project is to review the current state of the science of risk assessment of chemicals in food and diet, by consideration of the four stages of risk assessment, that is, hazard identification, hazard characterisation, exposure assessment and risk characterisation. The contribution of animal-based methods in toxicology to hazard identification of chemicals in food and diet is discussed. The importance of first applying existing technical and chemical knowledge to the design of safety testing programs for food chemicals is emphasised. There is consideration of the presently available and commonly used toxicity testing approaches and methodologies, including acute and repeated dose toxicity, reproductive and developmental toxicity, neurotoxicity, genotoxicity, carcinogenicity, immunotoxicity and food allergy. They are considered from the perspective of whether they are appropriate for assessing food chemicals and whether they are adequate to detect currently known or anticipated hazards from food. Gaps in knowledge and future research needs are identified; research on these could lead to improvements in the methods of hazard identification for food chemicals. The potential impact of some emerging techniques and toxicological issues on hazard identification for food chemicals, such as new measurement techniques, the use of transgenic animals, assessment of hormone balance and the possibilities for conducting studies in which common human diseases have been modelled, is also considered.

In order that a proposal for an alternative to an animal test be developed as an internationally accepted guideline, there needs to be consensus on the validity of the method proposed. Over the years, considerable attempts have been made to 'validate' promising alternatives. Probably without exception, these validation programmes demanded considerable budgets whereas the high expectations as to the output, which would justify the costs involved, were hardly ever met. What went wrong? Obviously, as for each new animal test, each new alternative to an animal test should be subjected to a critical appraisal procedure involving its scientific justification, its sensitivity and its reproducibility, before it could be internationally acceptable. Although there may be differences of opinion on the extent of this exercise, there is considerable agreement that validation in one way or another is essential. None the less, validation programmes so far have not resulted in the broad acceptance of any alternative test method. There may be two reasons for this failure. First, the results of the validation studies may have been unsatisfactory, which could mean that either the method subjected to validation failed to show the desired relevance and reliability, or the validation study as such yielded inconclusive results. Secondly, despite clear-cut (supporting) results from the validation exercise, toxicologists/regulators appear reluctant actually to use the data provided for hazard and risk assessment procedures because of a lack of confidence with the (types of) endpoints of the new test. The latter in particular can be considered a major hurdle in the process of acceptance of alternative tests. Therefore, an independent and objective review of any new test, with a view to its usefulness as a contribution to the set of data essential for hazard characterization and risk assessment, should be considered the first step of any comprehensive validation project. Further, the establishment of international centres such as the Johns Hopkins Center for Alternatives to Animal Testing (CAAT) and the European Centre for the Validation of Alternative Methods (ECVAM) where scientists, including regulators, can meet and discuss strategies not only for validation but also for the use of alternative methods in risk assessment, is considered essential for a good understanding of the relevance of the new in vitro, toxicity tests.

Toxicity studies are conducted with a view to identifying and assessing human or environmental hazards. Thus, the key issues are those of which, and how much, information is considered necessary to make a reliable and valid assessment, and to what extent alternative tests can contribute to this assessment. In this respect, the following fundamental aspects are of relevance. Toxicology as a science focuses on the identification and characterization of adverse effects following exposure to chemicals by integrating all available information, including physicochemical properties, all observed adverse effects in all studies, toxicokinetics and toxicodynamics, exposure conditions, and more. Most of this information is generated in tests conducted according to internationally accepted guidelines. Next, the art of toxicology is to evaluate and extrapolate the information acquired and then to assess human and environmental health hazards. Thus, the validity of any hazard prediction relies both on the collected data and on the craftsmanship of the assessor. Unfortunately, the experts involved in the science of toxicology are frequently not involved in the art of toxicology. As a consequence, the artist who is used to his set of fine tools, which he bought a long time ago and with which he always managed to create the 'sculpture' of hazard assessment, is now suddenly asked to use some new and rather different tools. It is understandable that this may bring about some opposition. In addition to these fundamental aspects, there are also political aspects to the issue of acceptance of alternative tests. These and other pitfalls have contributed to the author's opinion that progress in this respect can only be made by introducing the alternative tests without, at least for the time being, deleting the trusted existing ones.

The effects of prenatal oral administration of 0.2, 0.6, and 1.8 mg/kg body wt of 3,3',4,4'5,5'-hexachlorobiphenyl (HCB) on Day 1 of gestation and a combination of 1 mg/kg 3,3',4,4'-tetrachlorobiphenyl (TCB) from Day 2 to Day 18 with 0.6 mg HCB/kg body wt on Day 1 of gestation on thyroid hormone status and peripheral thyroid metabolism were studied in pregnant Wistar rats and their fetuses and offspring. Plasma total thyroxine and free thyroxine levels were reduced by HCB in a dose-dependent fashion in pregnant rats (Days 12 and 20 of gestation) and neonates (Day 21 postpartum), while only a combined dose of HCB and TCB was effective in decreasing fetal thyroid hormone levels by 65% on Day 20 of gestation. The activity of type II thyroxine 5'-deiodinase (5'D-II), the enzyme responsible for the deiodination of thyroxine (T4) to biologically active triiodothyronine in the brain, was examined in whole brain homogenates in fetuses and neonates. Decreases in plasma thyroid hormones were accompanied by significant increases, up to 100%, in 5'D-II activity in brain homogenates from fetuses (Day 20 of gestation) and neonates (Days 7 and 21 postpartum). The glucuronidation of 125I-T4 by hepatic microsomes was increased by at least 100% relative to control levels by all treatments in fetuses (Day 20 of gestation) and increased at least 40% in neonates (Days 7 and 21 postpartum) by a dose of 0.6 and 1.8 mg HCB/kg and the combined dose. These data indicate that prenatal HCB and/or TCB administration result in increased peripheral T4 metabolism. The increase in 5'D-II activity suggests that local hypothyroidism occurs in the brains of fetal and neonatal rats exposed to HCB and/or TCB. Since these effects occur during a period in which thyroid hormones play an important role in brain maturation, they may help explain the mechanism of developmental neurotoxicity induced by polychlorinated biphenyls.

Toxicity studies are necessary in order to be able to identify the potential hazards of chemical interference with human reproduction. Until today, most useful contributions to the assessment of possible human reproductive toxicity are considered to be made by animal studies. The OECD Test Guidelines provide the international standards for safety testing and, consequently, have traditionally focused on animal studies. However, the OECD Member countries consider the welfare of laboratory animals also of importance and are of the opinion that animal welfare considerations should significantly influence the work in the OECD Chemicals Programme. A constant effort is being made to discover alternative testing systems and to achieve their regulatory acceptance. However, activities predominately focus on finding alternatives to existing animal studies, rather than developing nonanimal tests that could contribute significantly to the hazard identification process. An approach based on the selection of endpoints essential for hazard identification that would focus on demonstrating similarities with the "real life" target events is considered likely to achieve regulatory acceptance much earlier than an approach based on high correlations between the alternative method and the existing animal study it is supposed to replace, simply because it is never better than the existing method, but at most "almost as reliable."

The enucleated eye test (EET) with the isolated eye of rabbits has been recognized as a valuable alternative to the Draize test, because it represents a test system nearest to the in vivo test, without the need to use live animals. In this ex vivo bioassay, three parameters are measured to detect possible adverse eye effects, namely corneal swelling, corneal opacity and fluorescein retention. The measurement of corneal swelling in this assay guarantees a highly objective and discriminative parameter. In combination with the detailed observation of corneal opacity and fluorescein retention, a reliable evaluation of the eye irritation potential of test materials is achieved. However, laboratory animals are still necessary as eye donors. The use of slaughter animals, such as the cow, the pig and the chicken, as possible as eye donors for the EET was therefore examined. From these candidates, the chicken appeared to be the most practicable. 21 reference compounds, ranging from non-irritant to severe irritant, which had been tested previously in a validation study on alternative test methods for eye irritation testing, sponsored by the Commission of the European Communities, were examined in the Chicken Enucleated Eye Test (CEET). When compared with the in vivo EC classification, the CEET correctly classified each of the compounds that must be labelled in the EC as irritant (R36) or severely irritant (R41). In addition, since the CEET recognizes three levels of irritancy rather than two (as in the case of the EC classification) a small number of the compounds were recognized as slightly irritant, which according to the EC classification need not be labelled. It was concluded that this ex vivo test system is highly accurate in the assessment of eye irritation potential without the use of laboratory animals.

Rabbit full thickness skin can be maintained viable for 7 days using a two-compartment organ culture model. We report that this model is useful for assessing cutaneous toxicity. 11 chemicals were each applied topically and then cytotoxicity was determined using two different assays. Mitochondrial activity was assessed by measuring reduction of the tetrazolium salt MTT, while uptake of the vital dye neutral red was used as a parameter of membrane damage. Conversion of MTT was inhibited in a dose-dependent way by all of the chemicals tested except dimethyl sulphoxide. Furthermore, when the skin explants were kept in culture after exposure to the test agents, both repair of initial damage and delayed toxicity could be observed to some extent. Uptake of neutral red was affected dose-dependently by six out of the 11 chemicals tested. The data indicate that a strong correlation exists between the cytotoxicity of chemicals and their effect on the conversion of MTT in skin organ culture. The MTT assay reported here offers the possibility of studying both acute and delayed cytotoxicity, and to some extent, recovery from initial damage.

An oral testicular toxicity and male fertility study was carried out in CD-1 mice with 1,3-diphenylguanidine (99.9% purity). 1,3-Diphenylguanidine was administered to male mice by daily gavage at dose levels of 0, 0.06, 0.25, 1, 4 and 16 mg/kg body wt. per day during an 8-week premating period. Females were not dosed at any time during the study. Sperm abnormality evaluation was performed in approximately half the males, randomly selected from the control and 16-mg/kg dose group on completion of dosing. The remaining males in the control, 4- and 16-mg/kg body wt per day groups were mated with non-dosed females. Reproductive performance, necropsy findings and litter data were recorded. No differences were found between control and dosed groups in body weight gain during the dosing period, macroscopic observations and organ weights at necropsy. Microscopic examination of the testes and determination of the frequency of total sperm abnormalities in the 16-mg/kg body wt per day group, did not show any effect due to 1,3-diphenylguanidine dosing when compared to the control group, except for a slight increase in sperm with folded tails but normal heads. Male and female fertility as well as reproduction performance were comparable in the groups examined (0, 4 and 16 mg/kg body wt per day). Maternal necropsy findings and litter data did not reveal any dose-related effect. It was concluded that under the conditions of the present study, 1,3-diphenylguanidine did not exert any significant adverse effects on fertility, reproductive capacity or embryonic/fetal development in CD-1 mice when administered to males at levels up to 16 mg/kg body wt per day.

A new method was developed for rabbit skin organ culture. In a two-compartment model, skin discs were cultured on a Millicell-HA insert unit with a microporous membrane which allows transport of culture medium via the dermis into the epidermis, whereas the epidermal side remains free of direct contact with culture medium. In this relatively simple two-compartment organ culture model, rabbit skin could be cultured for 7 d in RPMI 1640 medium supplemented with fetal bovine serum, or for 2 d in RPMI 1640 medium supplemented with cofactors. The histomorphology and ultrastructure of 7-d cultured rabbit skin discs was essentially similar to that of freshly isolated rabbit skin. Keratinocytes in the stratum basale continued to divide during organ culture. The terminal differentiation of the epidermis continued in vitro as was found by the presence of keratohyalin granules, the intact stratum corneum, and keratin expression. Furthermore, glucose consumption continued until culture Day 7, but thereafter it declined rapidly. Concomitantly, degenerative changes were found. At the end of the 7-d culture period the distance between single dermal collagen fibrils had increased as compared to noncultured skin. This model of skin organ cultures can be used to study biological processes, dermal toxicity, and penetration and metabolism of xenobiotics in intact skin. Furthermore, within certain limits, processes responsible for repair and regeneration of damaged skin can also be studied in this model because the rabbit skin can be cultured for 7 d.

The embryotoxicity/teratogenicity of the sugar replacer isomalt was studied in Wistar rats and New Zealand White rabbits. Groups of 22-23 female rats were given diets containing isomalt at concentrations of 2.5, 5 or 10%, from day 0 to day 21 of pregnancy. The possible adverse effects of restricted feeding were studied in an additional group (food intake less than 80% of the control values). Groups of 36-37 female rabbits were given diets containing isomalt at concentrations of 2.5, 5 or 10%, from day 0 to day 29 of pregnancy. The female rats and rabbits were killed on days 21 and 29 of pregnancy, respectively. In both species no maternal toxicity occurred and no effects on reproductive performance nor on embryonic or foetal development were seen in any of the groups fed isomalt. The feeding of restricted amounts of stock diet to pregnant rats resulted in decreased maternal weight gain and lower uterus weights. Furthermore, this group had an increased number of resorptions and small foetuses, decreased foetus and placental weights and retarded bone development.

The sugar replacer isomalt was fed to Wistar rats of both sexes throughout three successive generations at concentrations of 0, 2.5, 5 and 10% in the diet. A group of rats fed a diet containing 10% sucrose served as an additional control group. The initial mating was of 100 rats of each sex in each group. For subsequent matings 20 rats of each sex from each group were used. For each generation two litters were reared until they were at least 3 wk old. Feeding isomalt to rats for three successive generations did not induce any adverse effects on fertility, reproductive performance or development compared with control animals fed diets containing maize starch and sucrose instead of isomalt. The second litter of third-generation rats was given detailed gross and microscopic examinations 4 wk after weaning. A marked treatment-related change was an increase in the relative weight of the caecum (filled and empty), 4 wk after weaning in the second litter of third-generation rats fed 10% isomalt. There was also an increase in the relative weight of the filled caecum in males of the 5% isomalt group. These findings were not accompanied by diarrhoea or histological changes in the caecum. The results of the present study did not demonstrate any deleterious effects on the reproduction, maternal performance or development of rats fed isomalt at dietary levels of up to 10% over three successive generations.

Mice exposed to 6 hours of 75% N2O or 0.5% halothane on the 14th day of gestation and mice exposed to 4 hours of 75% N2O or 0.5% halothane on the second day of life were tested for general activity at 3 ages. Just before weaning, all treated groups appeared hypoactive and there was a significant effect in the postnatal nitrous oxide group. Both postnatal groups had deviant distributions of scores, compared to sham-exposed controls. The main effect of treatment was greater in young adulthood, when the prenatal halothane group differed from controls. This group and the postnatal groups had excess numbers of low-scoring animals. At six months, the main effect of treatment was no longer significant, but the unusual distribution of scores was still present in the postnatal halothane group. Correlations of individual scores over the whole experiment suggested that early activity measures were significantly related to later scores. Within treatment groups, controls and postnatal nitrous oxide animals were the most consistent over time. Both agents appear to alter activity levels for periods too long to be explained by lingering presence of the drug or its metabolities.

Toxicity data of 37 compounds tested in both a subchronic study and one or more studies focused on reproduction were evaluated to obtain information on the significance of fertility and reproduction parameters in setting no-effect levels (NEL). It appeared that a) the test system aimed at reproduction and development was more sensitive than the subchronic study for 35% of the selected compounds and was equally sensitive for another 35%; b) for 65% of these compounds parameters related to reproduction and development were at least codecisive in determining the minimal-effect level (MEL); c) for 21% of all compounds, parameters related to reproduction were the most sensitive and therefore decisive for the MEL. It was concluded that reproduction in its broadest sense is a very sensitive tool in toxicity testing. The possibilities of either applying a simple screen of fertility and reproduction at an early stage of toxicity testing or incorporating fertility and reproduction parameters in the current subchronic study are discussed.

Mice exposed to four or six hours of nitrous oxide or halothane differed from controls on a variety of tests conducted before weaning. Whereas many agents that produce behavioral terata have very different effects at different stages of brain development, these inhalant anesthetics had similar effects, whether exposure occurred on the 14th day of gestation or two days after birth. Both treatment times and both agents were associated with delays in the appearance of developmental landmarks and delays in the appearance of righting reflexes and locomotion. The level of general activity just before weaning tended to be low in all treated groups and was significantly depressed in males exposed to N2O postnatally. The distribution of activity scores was shifted significantly in both postnatal groups compared to controls. The data are compatible with human studies suggesting that inhalants at parturition have an effect on early behavior. The persistence of effects over the first three weeks of life does not fit with the idea that the behavioral effects are mediated by continued presence of the drug. The similar effects of the two agents, which produce very different degrees of anesthesia, supports earlier studies suggesting that the teratogenicity of inhalants is independent of the level of anesthesia produced.

To study the role of particle size of benzo(a)pyrene (BP) in the induction of respiratory tract tumours, goups of hamsters were given 52 weekly intratracheal instillation of fine (77% less than 5.2 micron; 3% less than 1.3 micron) or coarse (77 less than 42 micron; 0% less than 10 micron) BP particles suspended in gelatin-0.9% NaCl solution. Both the fine and coarse particles were given at two different dose levels, viz., 0.5 and 1.0 mg BP/instillation. Another group of hamsters received 52 weekly intratracheal instillations each consisting of 1.0 mg BP particles of a wide size range (90% less than 50 micron; 3% less than 5.0 micron) suspended in 0.9% NaCl solution without gelatin. The retention of BP in the respiratory tract was studied in separate groups of hamsters receiving a single instillation of BP particles of the different size ranges. Coarse BP particles cleared less rapidly from the lungs and induced a much stronger tumour response in the respiratory tract than did fine BP particles. These findings demonstrated the crucial significantce of particle size for the effectiveness of BP as a respiratory tract carcinogen. No appreciable differences in site of tumour formation were observed between treatment with fine and coarse BP particles, which led to the conclusion that the size of the particles did not play an important role in determining the location of the induced respiratroy tract tumours. The elimination rate of the BP particles with a wide size range appeared to be in between those of the fine and the coarse particles. The tumour response found after administration of the BP particles of wide size range was very similar to that obtained with the high dose of coarse PB particles.

Several inhalant anesthetics, including nitrous oxide and halothane, are known to be antimitotic in a variety of developing tissues, but none has been tested for antimitotic activity in developing brain. Concern about the safety of these agents has centered around behavioral effects reported in humans and animals after early exposure. Because interference with cell production during CNS development is a sufficient cause for later behavioral abnormalities, it is important to know whether cell production in the nervous system is altered by these agents. Mice were exposed to either nitrous oxide (75% N2O and 25% O2) or halothane (0.5% halothane in 75% N2 and 25% O2) or a mixture of 75% N2 and 25% O2. Prenatal treatment groups were exposed for 6 h on the 14th day of gestation, while postnatal treatment groups were exposed for 4 h on the second day after birth. Treated and control animals were then killed immediately after exposure, or 12, 24, or 48 h later, to be evaluated for CNS mitotic activity. Each of the four anesthetic-exposed groups showed some deviations from normal mitosis, but only the postnatal nitrous oxide group showed the pattern of reduced cell proliferation followed by a rebound that is characteristic of many antimitotic teratogens. Although prenatal nitrous oxides' effects on the fetal brain were not clearly interpretable, it did delay development of blood, as has been reported by other investigators. Both nitrous oxide and halothane significantly reduced body weight of fetuses in utero, but did not reduce body weight of neonates. The pattern of the body-weight effects suggests that they occur by some mechanism other than reduced cell production.(ABSTRACT TRUNCATED AT 250 WORDS)

The sugar replacer isomalt, a 1:1 mixture of the disaccharides glucopyranosylsorbitol and glucopyranosylmannitol, was incorporated in the diet of rats. Female Bay FB:30 rats were adapted to isomalt by feeding them a diet containing a gradually increasing amount of isomalt for several days, prior to mating. Subsequently, they were mated. Isomalt was fed continuously in concentrations of 2.5, 5 and 10% up to day 20 of pregnancy. In addition, one group of female Wistar rats was mated and fed 10% isomalt incorporated in the diet from day 0 up to day 20 of pregnancy, without previous adaptation to isomalt. Finally, one group of untreated female Wistar rats served as controls. Half of the number of females in each group was selected for caesarian section on day 20 of pregnancy. The other half was allowed to litter and rear their pups for 2 weeks (Wistar rats) or 3 weeks (Bay FB:30 rats). In the females of the Bay FB:30 rats, a decreased body-weight gain and food consumption were observed in the 5 and 10% isomalt group. Minor retardation in the development of the foetuses was observed in the 10% isomalt group only with the Bay FB:30 rats and was therefore considered to be related to maternal toxicity. In addition, a dose-related increase in the incidence of wavy ribs occurred in foetuses of the Bay FB:30 rats. However, none of the observed effects were persistent in neonates. Isomalt appeared to have slight toxic effects in the dams of the Bay FB:30 strain but no toxicity in the offspring. In Wistar rats no toxicity and no effects on maternal performance or on embryonic, foetal or neonatal development were seen. Isomalt, when fed at dietary levels up to 10%, did not induce structural or functional teratogenic effects in rats of either the Wistar or the Bay FB:30 strain.