25th anniversary of the Berlin workshop on developmental toxicology: DevTox database update, challenges in risk assessment of developmental neurotoxicity and alternative methodologies in bone development and growth.

25 years after the first Berlin Workshop on Developmental Toxicity this 10th Berlin Workshop aimed to bring together international experts from authorities, academia and industry to consider scientific, methodologic and regulatory aspects in risk assessment of developmental toxicity and to debate alternative strategies in testing developmental effects in the future. Proposals for improvement of the categorization of developmental effects were discussed as well as the update of the DevTox database as valuable tool for harmonization. The development of adverse outcome pathways relevant to developmental neurotoxicity (DNT) was debated as a fundamental improvement to guide the screening and testing for DNT using alternatives to animal methods. A further focus was the implementation of an in vitro mechanism-based battery, which can support various regulatory applications associated with the assessment of chemicals and mixtures. More interdisciplinary and translation research should be initiated to accelerate the development of new technologies to test developmental toxicity. Technologies in the pipeline are (i) high throughput imaging techniques, (ii) models for DNT screening tests, (iii) use of computer tomography for assessment of thoracolumbar supernumerary ribs in animal models, and (iv) 3D biofabrication of bone development and regeneration tissue models. In addition, increased collaboration with the medical community was suggested to improve the relevance of test results to humans and identify more clinically relevant endpoints. Finally, the participants agreed that this conference facilitated better understanding innovative approaches that can be useful for the identification of developmental health risks due to exposure to chemical substances.

Safety assessment of a novel C-type natriuretic peptide derivative and the mechanism of bone- and cartilage-specific toxicity.

ASB20123, a C-type natriuretic peptide/ghrelin chimeric peptide, was designed as a novel peptide and demonstrated full agonistic activity for natriuretic-peptide receptor B and a significantly longer half-life in plasma compared with the native peptide. We researched the toxicological profile of ASB20123, the correlation between the morphological change of the epiphyseal plate and bone and cartilage toxicity, and biomarkers to detect the toxicity. ASB20123 was systemically administered to male and female rats at daily dose levels of 0.5, 1.5, and 5.0 mg/kg/day for 4 weeks. In this study, toxicity was observed as changes related to bone and cartilage tissues, and no other toxicological changes were observed in all animals. Next, ASB20123 was administered to 12-month-old rats with a little epiphyseal plate. The toxic changes related to bone and cartilage tissues were not observed in any animal with a closed epiphyseal plate, indicating that the toxic changes were triggered by the growth-accelerating effect on the bone and cartilage. Furthermore, we searched for the biomarker related to the bone and cartilage toxicity using rats treated with ASB20123 at doses of 0.005, 0.05, 0.5, and 5.0 mg/kg/day for 4 weeks. A close correlation between necrosis/fibrosis in the epiphysis and metaphysis and thickness of the epiphyseal plate in the femur was confirmed in this study. A decrease in the bone mineral density (BMD) of the femur also was associated with the appearance of bone toxicity. These results indicated that the toxicity of ASB20123 was limited to bone- and cartilage-specific changes, and these changes were triggered by an excessive growth accelerating effect. Furthermore, our data suggested that the thickness of the epiphyseal plate and BMD could be reliable biomarkers to predict bone toxicity.

Calvarial Model of Bone Augmentation in Rabbit for Assessment of Bone Growth and Neovascularization in Bone Substitution Materials.

The basic principle of the rabbit calvarial model is to grow new bone tissue vertically on top of the cortical part of the skull. This model allows assessment of bone substitution materials for oral and craniofacial bone regeneration in terms of bone growth and neovascularization support. Once animals are anesthetized and ventilated (endotracheal intubation), four cylinders made of polyether ether ketone (PEEK) are screwed onto the skull, on both sides of the median and coronal sutures. Five intramedullary holes are drilled within the bone area delimited by each cylinder, allowing influx of bone marrow cells. The material samples are placed into the cylinders which are then closed. Finally, the surgical site is sutured, and animals are awaken. Bone growth may be assessed on live animals by using microtomography. Once animals are euthanized, bone growth and neovascularization may be evaluated by using microtomography, immune-histology and immunofluorescence. As the evaluation of a material requires maximum standardization and calibration, the calvarial model appears ideal. Access is very easy, calibration and standardization are facilitated by the use of defined cylinders and four samples may be assessed simultaneously. Furthermore, live tomography may be used and ultimately a large decrease in animals to be euthanized may be anticipated.

A shortened study design for embryo-fetal development studies in the minipig.

The minipig is accepted from scientific and regulatory perspectives for the safety evaluation of drug candidates on embryo-fetal development. The relative size and the duration of gestation (112-115 days) in the minipig is, however, considered a drawback compared with routine smaller species. We evaluated if study duration and cost could be optimized without impacting scientific validity by performing all terminal procedures around mid-gestation (60 days). At this stage, minipig fetal size is not too dissimilar to full term rabbit and therefore better suited to fetal processing/examination compared with at the end of gestation. Despite encountering higher than anticipated embryo-fetal death, morphological defects clearly associated with a known teratogen, pyrimethamine, were detected. Although the gonads are poorly differentiated macroscopically at mid-term, a histological examination confirmed that external sexing of the fetuses was accurate. Double staining of the bone and cartilage of the mid-term fetal skeleton allowed a more refined examination.

A 4-Year, Open-Label, Multicenter, Randomized Trial of Genotropin® Growth Hormone in Patients with Idiopathic Short Stature: Analysis of 4-Year Data Comparing Efficacy, Efficiency, and Safety between an Individualized, Target-Driven Regimen and Standard Dosing.

BACKGROUND/AIMS: Growth hormone (GH) treatment regimens for children with non-GH-deficient, idiopathic short stature (ISS) have not been optimized. To compare the efficacy, efficiency, and safety of an individualized, target-driven GH regimen with standard weight-based dosing after 4 years of treatment. METHODS: This is a 4-year, open-label, multicenter, randomized trial comparing individualized, formula-based dosing of Genotropin® versus a widely used ISS dose of Genotropin®. Subjects were prepubertal, had a bone age of 3-10 years for males and 3-9 years for females, were naive to GH treatment, and had a height standard deviation score (Ht SDS) of -3 to -2.25, a height velocity <25th percentile for their bone age, and peak stimulated GH >10 ng/ml. After the first 2 years, the individualized-dosing group was further randomized to either 0.18 or 0.24 mg/kg/week. RESULTS: At 4 years, subjects in all treatment regimens achieved similar average height gains of +1.3 SDS; however, the individualized dosing regimen utilized less GH to achieve an equivalent height gain. CONCLUSION: Individualized, formula-based GH dosing, followed by a dose reduction after 2 years, provides a more cost-effective growth improvement in patients with ISS than currently employed weight-based regimens.

Assessment of bone health in children with disabilities.

Evaluating the bone health of children with disabilities is challenging and requires consideration of many factors in clinical decision-making. Feeding problems and growth deficits, immobility/inability to bear weight, effect of medications, and the nature of his or her disease can all directly affect a child's overall picture of bone health. Familiarity with the tools available to assess bone health is important for practitioners. The most commonly used method to assess bone density, dual energy x-ray absorptiometry, can be performed effectively when one appreciates the techniques that make scanning patients with disabilities possible. There are specific techniques that are especially useful for measuring bone density in children with disabilities; standard body sites are not always obtainable. Consideration of clinical condition and treatment must be considered when interpreting dual energy x-ray absorptiometry scans. Serial measurements have been shown to be effective in monitoring change in bone content and in providing information on which to base decisions regarding medical treatment.

Assessment of a nonsteroidal aromatase inhibitor, letrozole, in juvenile rats.

BACKGROUND: The timing and duration of letrozole administration was designed to encompass the majority of postnatal development in the rat with the intent of evaluating the potential for a broad range of effects but with emphasis on expected effects on skeletal maturation. METHODS: Sprague-Dawley rats were administered letrozole via oral gavage at doses of 0.003, 0.03, and 0.3 mg/kg/day beginning on postpartum day (PPD) 7 through 91 followed by a 6-week recovery period. Clinical signs, body weight, food consumption, developmental endpoints, bone, ophthalmology, behavioral assessments, clinical/anatomic pathology, toxicokinetics, and reproductive assessments were conducted. RESULTS: Growth (body weight gain and crown-to-rump length) and food consumption were increased in females at ≥0.03 mg/kg/day and decreased in males at ≥0.003 mg/kg/day. Delayed sexual maturation in both sexes and adverse effects on reproductive function occurred at all doses. Effects on bone growth and maturation were noted in both sexes at all doses. Evidence of recovery was noted for males at 0.003 mg/kg/day and females at 0.003 and 0.03 mg/kg/day upon withdrawal of treatment. Histopathological changes in the pituitary-adrenal-gonadal axis correlated with effects on reproductive function. CONCLUSIONS: The observed effects in juvenile rats were considered predictable and primarily related to the mechanism of action of letrozole upon estrogen synthesis.

Growth-attenuation therapy: principles for practice.

Publication of an account of growth attenuation with high-dose estrogen in a child with profound physical and cognitive disability brought widespread attention to a common and complex issue faced by families caring for similarly affected children, namely, the potentially negative effect of the increasing size of a child on the ability of his or her family to provide independent care, which in turn makes it more difficult for parents to keep the child in the home and involved in family activities. In this article we explore the scientific rationale for, effectiveness and safety of, and ethical considerations bearing on growth-attenuation treatment of children with profound and permanent cognitive disability. Informed responses to key clinically relevant questions are proposed. Our analysis suggests that growth attenuation is an innovative and sufficiently safe therapy that offers the possibility of an improved quality of life for nonambulatory children with profound cognitive disability and their families. Pediatricians and other care providers should include discussion of these options as part of anticipatory guidance around the age of 3 years so that, if elected, potential clinically meaningful benefits of growth-attenuation therapy can be realized. Because of the publicity and debate surrounding the first reported case, ethics consultation is recommended.

Effects of school milk intervention on cortical bone accretion and indicators relevant to bone metabolism in Chinese girls aged 10-12 y in Beijing.

BACKGROUND: We previously reported that increased milk consumption enhances growth and bone mineral accretion in Chinese girls aged 10-12 y. OBJECTIVE: Our objective was to evaluate the effects of milk supplementation on cortical bone accretion and to study the physiologic mechanisms underlying the observed changes in bone. DESIGN: Chinese girls aged 10 y were randomly assigned into calcium-fortified milk (Ca milk), calcium and vitamin D-fortified milk (CaD milk), and control groups according to their schools in a 24-mo school milk intervention trial. Periosteal and medullary diameters of metacarpal bone were measured at baseline and 24 mo in the Ca milk (n = 177), CaD milk (n = 210), and control (n = 219) groups. Insulin-like growth factor I (IGF-I), parathyroid hormone (PTH), bone alkaline phosphatase (BAP), osteocalcin, and deoxypyridinoline concentrations were measured at baseline and at 12 and 24 mo in the Ca milk (n = 43), CaD milk (n = 44), and control (n = 41) groups. RESULTS: After adjustment for pubertal status and clustering by school, 24-mo supplementation led to greater increases in periosteal diameter (1.2%) and cortical thickness (5.7%) and to smaller gains in medullary diameter (6.7%) than did the control (P < 0.05). The CaD milk group had lower serum BAP at 12 mo (19.9%) and lower serum PTH at 12 (46.2%) and 24 (16.4%) mo than did the control group (P < 0.05). The effect of milk supplementation on increasing IGF-I concentrations at 24 mo (16.7-23.3%) was significant in individual analyses but not after adjustment for clustering by school. CONCLUSIONS: Milk supplementation showed positive effects on periosteal and endosteal apposition of cortical bone.

Assessment of the perinatal effects of maternal ingestion of Solanum malacoxylon in rats.

A perinatal study was performed to verify the toxic effects of Solanum malacoxylon, which contains a glycoside conjugated to Vitamin D(3). In the gestational study, female rats received S. malacoxylon leaves in the diet at 0, 0.1, 0.2, 0.5, and 1% from days 6 to 21 of pregnancy. At 21 days of gestation, blood samples were taken from the dams for evaluation of serum Ca and P. A laparotomy was performed and the rats were examined for standard parameters of reproductive performance. Fetuses were examined for skeletal changes and histopathologic evaluation. In the second trial, dams were fed diets containing 0 or 0.1% S. malacoxylon leaves during the gestation and lactation periods. After weaning, all animals were euthanized and biochemical and histopathologic evaluations were performed. The biochemical evaluation showed increase in Ca and P levels in females from all experimental groups; however, this effect did not occurred in a dose-related manner. Pups from dams exposed during gestation and lactationi also showed increased Ca and P levels. Fetal data suggested a delay of fetal development manifested by decreased body weight and skeletal alterations. There was also a reduction in live fetuses. Histopathologic study revealed alterations of the soft tissue in litters from dams given 1% dietary S. malacoxylon during pregnancy and 0.1% during pregnancy and lactation. These findings support our hypothesis that Vitamin D(3) glycoside crosses the placenta and suggests milk transfer of this substance.

High dose growth hormone treatment induces acceleration of skeletal maturation and an earlier onset of puberty in children with idiopathic short stature.

BACKGROUND: Long term growth hormone (GH) treatment in children with idiopathic short stature (ISS) results in a relatively small mean gain in final height of 3-9 cm, which may not justify the cost of treatment. As it is unknown whether GH treatment during puberty adds to final height gain, we sought to improve the cost-benefit ratio, employing a study design with high dose GH treatment restricted to the prepubertal period. AIMS: To assess the effect of short term, high dose GH treatment before puberty on growth, bone maturation, and pubertal onset. METHODS: Five year results of a randomised controlled study are reported. Twenty six boys and nine girls were randomly assigned to a GH treatment group (n = 17) or a control group (n = 18). Inclusion criteria were: no signs of puberty, height less than -2 SDS, age 4-8 years for girls or 4-10 years for boys, GH concentration >10 micro g/l after provocation, and normal body proportions. To assess GH responsiveness, children assigned to the GH treatment group received GH treatment for two periods of three months (1.5 IU/m2/day and 3.0 IU/m2/day), separated by three month washout periods, during the first year of study. High dose GH treatment (6.0 IU/m2/day) was then started and continued for at least two full years. When puberty occurred, GH treatment was discontinued at the end of a complete year's treatment (for example, three or four years of GH treatment). RESULTS: In response to at least two years on high dose GH treatment, mean (SD) height SDS for chronological age increased significantly in GH treated children from -2.6 (0.5) to -1.3 (0.5) after two years and -1.4 (0.5) SDS after five years of study. No changes in height SDS were observed in controls. A rapid rate of bone maturation of 3.6 years/2 years in treated children compared to 2 years/2 years in controls was observed in response to two years high dose GH treatment. Height SDS for bone age was not significantly different between groups during the study period. GH treated children entered into puberty at a significantly earlier age compared to controls. CONCLUSIONS: High dose GH treatment before puberty accelerates bone age and induces an earlier onset of puberty. This may limit the potential therapeutic benefit of this regimen in ISS.

Methodological aspects of short-term knemometry in the assessment of exogenous glucocorticosteroid-induced growth suppression in children.

During recent years knemometry has been introduced for short-term assessment of the growth-suppressive effect of exogenous glucocorticosteroids in children. The aim of the present paper is to review methodological aspects of short-term knemometry used for this purpose. Knemometry has proven a highly accurate and reproducible method for assessment of short-term growth suppression in populations of children treated with exogenous glucocorticosteroids. Randomized, double-blind crossover and parallel designs applying consistent measurement intervals can be used. Confounding influences on the growth results from possible inter-group differences in spontaneous growth velocities are reduced in the crossover design. Glucocorticosteroid-induced knemometric growth suppression seems to reflect suppressive effects on soft tissue and bone components in the lower leg. In children treated with systemic glucocorticosteroids a shortening of the lower leg, which may be due to a reduction of the water content in the soft tissue, may confound the growth assessment. Suppressed short-term growth rates should be considered to have a poor correlation with long-term growth, though prospectively planned, controlled studies of the relation are needed in glucocorticosteroid-treated children.

Longitudinal assessment of bone mineral density in children with chronic asthma.

BACKGROUND: With the emphasis on asthma as a chronic inflammatory process, the management of moderate to severe asthma, even in the pediatric population, has shifted to the regular use of inhaled anti-inflammatory agents, including inhaled corticosteroids. Accompanying the use of these agents has been the precaution that long-term use may have subtle or potential side effects, including growth suppression or decreased bone mineral deposition. OBJECTIVE: We sought to study the effects of inhaled anti-inflammatory agents on bone mineral density accumulation in growing asthmatic children. Included in this report is the longitudinal acquisition of bone mineral density in children with moderate to severe asthma. METHODS: Bone mineral density in normal and asthmatic children was measured longitudinally by dual-energy absorptiometry. Bone densitometry was determined twice over a 7- to 16-month period in 21 asthmatic children and a 13- to 60-month period in 14 normals. These children with two longitudinal visits were compared with a group of 107 normal children who had a single bone mineral density measurement. RESULTS: Nineteen of 21 asthmatic children used regular inhaled corticosteroids during the interval visits. The majority of the asthmatic boys had bone mineral density measurements, at both visits, that were at a higher percentile than normal boys with two visits. Asthmatic girls had bone density measurements at percentiles not significantly different than normal girls with two visits. CONCLUSIONS: The advancement of bone mineral density in asthmatic children provides support for the safety of inhaled anti-inflammatory medications on bone mineral density in children with significant asthma.

A review of calcium preparations.

There are more than a dozen commonly prescribed calcium supplements and hundreds of different formulations commercially available. Numerous factors need to be considered when selecting a calcium preparation. Physical properties such as solubility, interference from coingested medications or foodstuffs, dosage, and timing can all affect the bioavailability of calcium. Medical conditions such as lactose intolerance, impaired gastric acid secretion, and high risk profile for kidney stone formation may impact on selection of a calcium supplement. This article will review the available literature and make general recommendations for the optimal use of calcium preparations.

Safety assessment of hydrogenated starch hydrolysates.

Hydrogenated starch hydrolysates (HSH) are mixtures of polyhydric alcohols such as sorbitol, maltitol, and higher-order sugar alcohols. They are important food ingredients because of their sweetness, low cariogenic potential, and useful functional properties. These traits permit HSH products to be used as viscosity or bodying agents, humectants, crystallization modifiers, and rehydration aids. A substantial body of safety information is available for HSH products and their individual chemical components. Based on this information, the substances have received favorable evaluations from international expert safety organizations such as the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and European Community's Scientific Committee for Food. This same information has been submitted to the United States Food and Drug Administration (FDA) as part of the petitioning process to affirm the generally recognized as safe (GRAS) status of these substances. Some of the animal feeding studies important to a full safety assessment for HSH substances, while long available to international safety expert organizations and governmental organizations, have never been published in the literature. Three of these studies, i.e., a chronic (24-month) feeding study, a multigeneration reproduction study, and a teratology study, are reported on this article, together with metabolic information. The results of this evaluation establish HSH substances as safe food ingredients.

Histomorphometric assessment of the mechanisms for rapid ingrowth of bone to HA/TCP coated implants.

The purpose of this work is to use dynamic histomorphometry to evaluate the basic biological mechanisms by which hydroxyapatite/tricalcium phosphate (HA/TCP) implant coatings accelerate bone formation rates. Twenty-five rabbits had an HA/TCP coated cylindrical titanium fiber metal mesh implant surgically placed in the subchondral bone of the proximal tibia and a noncoated implant placed in the contralateral tibia. Twenty-two of these animals had HA/TCP coated cylindrical solid titanium implants placed in the distal femur and an uncoated implant placed in the contralateral femur. The animals were double labeled with vital stains, and sacrificed at 3, 6, 16, or 26 weeks after surgery. Histomorphometric analyses were done of the bone implant interfaces. Both static and dynamic histomorphometric parameters indicate that HA/TCP coatings stimulate faster bone ingrowth to coated fiber metal implants through the early production of woven bone and by subsequent rapid lamellar bone formation rates. Coated fiber metal implants demonstrated significantly more bone ingrowth than noncoated implants through 16 weeks postimplantation, but not by 26 weeks. In solid implants, the differences between coated and noncoated implants are less pronounced and not statistically significant, although there is a trend toward increased bone apposition to the surface of the implants over the first 16 weeks following implantation. The clinical significance of these results is that coated implants may allow earlier return to normal weightbearing.

Simultaneous assessment of bone resorption and formation in cultures of 22-day fetal rat parietal bones: effects of parathyroid hormone and prostaglandin E2.

We have developed a method that allows us to measure bone resorption and formation simultaneously in the parietal bones from 22-day fetal rat calvaria. Parietal bones labeled with 45Ca, by injection of the mother, were cultured for 72 h with parathyroid hormone (PTH, bovine 1-34, 1.56 nM) or prostaglandin E2 (PGE2, 100 nM), in the presence or absence of indomethacin (Indo, 1 microM) or corticosterone (Cort, 1 microM). Two hours prior to the end of the culture, the bones were pulsed with [3H]-proline or [3H]-thymidine. Resorption was assessed as the percent of 45Ca released into the medium. Incorporation of [3H]-proline into collagenase digestible protein (CDP) and of [3H]-thymidine into DNA (TDR) were measured to assess collagen and DNA synthesis, respectively. Basal %45Ca release was 16 +/- 1% and was significantly decreased by Indo and Cort. Cort decreased TDR and CDP while Indo did not. PTH and PGE2 significantly increased %45Ca release, and this was not blocked by Indo. However, in the presence of Cort, only PTH increased %45Ca release while PGE2 did not. PGE2 increased TDR under all culture conditions while PTH increased TDR only in the presence of Cort. While PTH and PGE2 had the same effects on bone resorption, they had different effects on CDP. PGE2 increased CDP in the presence of Indo or Cort but PTH did not. Thus, this model allows us to study bone resorption, collagen synthesis, and DNA synthesis simultaneously. We have also shown that PTH and PGE2 differ in their sensitivity to inhibition of resorption by Cort and in their effects on bone formation.

Short children, anxious parents: is growth hormone the answer?

KIE: The impending Food and Drug Administration approval of a synthetic growth hormone (hGH) for use with children deficient in the hormone is expected to present a dilemma for physicians confronted by parental demands for hGH treatment of normal, but short, children. Little is known of long term effects of such treatment, which is protracted, slow to yield results, costly, and likely to cause psychological problems when ineffective. Public policy concerns include treating normal people to attain an ideal goal, equity in distribution of a costly treatment, reinforcement of "heightism," and the justifiability of clinical research on non-hGH deficient children.^ieng