Safety evaluation of phytosterol esters. Part 8. Lack of genotoxicity and subchronic toxicity with phytosterol oxides.

Vegetable oil spreads containing phytosterol-esters are marketed as a cholesterol-lowering functional food in more than 20 countries worldwide. An extensive package of safety data has shown phytosterol-esters to be safe for human use. However, even though phytosterols are very stable molecules, oxidation may occur at low levels under extreme heating conditions, resulting in phytosterol oxides. As there is some suggestion of adverse biological effects in the literature for the related cholesterol oxidation products, safety data have been generated for phytosterol oxides. A phytosterol oxide concentrate (POC) was generated by prolonged heating of phytosterol-esters in the presence of oxygen. The genotoxicity and subchronic toxicity of this mixture was assessed in a series of in vitro genotoxicity assays (bacterial mutation, chromosome aberration and micronucleus) and a subchronic feeding study in the rat. Results showed that a phytosterol oxide concentrate containing approximately 30% phytosterol oxides did not possess genotoxic potential and no obvious evidence of toxicity when administered in the diet of the rat for 90 consecutive days. In the latter study, a NOEL was established at an estimated dietary level of phytosterol oxides of 128 mg/kg/day for males and 144 mg/kg/day for females. In conclusion, these materials have been shown to raise no obvious concerns for human safety.

Safety evaluation of a glutaraldehyde modified tyrosine adsorbed housedust mite extract containing monophosphoryl lipid A (MPL) adjuvant: a new allergy vaccine for dust mite allergy.

A new allergy vaccine is currently under clinical evaluation for the prevention or relief of symptoms caused by specific housedust mites. It consists of a 50:50 mixture of the mite Dermatophagoides pteronyssinus and D. farinae protein derived from aqueous extracts of the mites which is chemically modified by glutaraldehyde and adsorbed onto L-tyrosine with addition of the immunostimulatory adjuvant, monophosphoryl lipid A (MPL) "Polymite". A specific preclinical safety testing strategy was developed to support clinical use and comprised single and repeat dose toxicity, reproduction toxicity and local tolerance studies. Dose levels of up to 0.5ml for the mouse and up to 1ml for both the rat and the rabbit were used. Overall, the product was shown to produce no toxicological findings of significance at levels greatly in excess to those proposed for clinical use. A not unexpected, but relatively minor, immunostimulatory effect was seen following repeated dosing (once weekly for 13 weeks) at 1ml per rat; the Polymite formulation also resulted in injection site reaction which can largely be attributed to the presence of tyrosine. No reproduction toxicity was found.

Reproduction studies in the rat with shea oleine and hardened shea oleine.

Shea oleine is an oil fraction derived from the nut of the tree Butyrospermum parkii, which grows in central and western Africa. There are several uses of shea oleine including its use as a frying oil and, after hardening, in margarine and toffee fat. This investigation was performed to examine the toxicity of 7 or 15% hardened shea oleine in comparison with 7 or 15% unhardened shea oleine and various commercially available materials, sheanut and palm oils, cocoa butter and toffee powder following dietary administration to rats during pre-mating, mating, pregnancy and offspring weaning in two separate investigations. Reproduction was assessed using number of litters and pups born plus survival and body weights at birth and at weaning on day 21. Skeletal evaluation using X-ray, clinical pathology and a macroscopic examination were also performed for F1 rats. Study measures for parent animals comprised evaluation of body weight, food consumption, clinical pathology, organ weights and macroscopic examination. Fatty acids and hydrocarbon levels were measured and an evaluation for lipogranulomata was made for various tissues. Results showed that shea oleine, whether unhardened or hardened, produced no evidence of reproduction toxicity and gave a similar profile to the other commercially available materials used in this study in the rat. Minor findings with shea oleine were not related to reproduction performance but comprised slightly reduced body weight gain and reduced cholesterol and raised alkaline phosphatase levels. None of the findings in this study were considered to be of toxicological significance. Thus, no evidence of reproduction toxicity was seen for both unhardened and hardened shea oleine in this investigation in the rat at levels equating to greater than 7.5 g/kg/day.

An assessment of the carcinogenic potential of shea oleine in the rat.

Shea oleine, an oil fraction derived from the nut of the tree Butyrospermum parkii, is used as a frying oil. As part of a series of studies, this investigation examined the carcinogenic potential of 15% (w/w) shea oleine in comparison with 15% (w/w) sheanut oil, and palm oil following dietary administration to rats over 104 weeks. The assessment comprised an evaluation of mortality, clinical signs, body weight, food intake, clinical pathology, organ weights and macroscopic and histopathological examination plus tumour type and incidence evaluation. Results showed that shea oleine produced no adverse effects and no evidence of tumorigenic potential compared to other commercially available sheanut and palm oils in the rat. Notable differences were confined to reduced body weight gain and food intake, reduced cholesterol and increased alkaline phosphatase levels, reduced heart weight and an increased incidence of pulmonary lipidosis with shea oleine diets. The latter effect may reflect a naturally lower incidence of this finding with palm oil diets. Tumour findings, specific to shea oleine diets, were restricted to an increase in the number of hepatomas for females, pancreatic exocrine adenomas for males and skin keratoacanthomas for males fed shea oleine diets. The increase in the incidence of hepatomas with treatment was thought to be related to the high fat content of the diets. The incidence of these tumour findings was similar to that given in published data for the Wistar rat, or the 'in house' values for tumour incidence in rats fed high-fat diets. In conclusion, none of the findings in this study were considered to be adverse effects. In comparison with other commercially available edible oils, shea oleine showed no tumorigenic potential following dietary administration at 7.5 g/kg/day in the rat.

Rubidium reduces potassium permeability and fluid secretion in Malpighian tubules of Locusta migratoria, L.

The basal membrane potential (V(b)) of Locusta Malpighian tubule cells in control saline results from its relatively high permeability to potassium. In the presence of 1 mM barium added to the control saline V(b) hyperpolarized from a mean resting potential of -72.1 mV to -90.1 mV. On substituting rubidium for potassium in the control saline, V(b) also hyperpolarized to a value of -91.4 mV. Rubidium was also similarly effective in hyperpolarizing the basal membrane even in the presence of control concentrations of potassium in the bathing medium. Substitution of rubidium for potassium also effected a approximately 50% reduction in the rate of fluid secretion. The action of inhibitors on V(b) in the presence of rubidium showed that V(b) under these conditions probably originated from the bafilomycin-sensitive electrogenic potential generated across the apical membrane by a V-type ATPase. The responses of V(b) to potassium, barium and rubidium and their inhibition of fluid secretion suggest the presence of a substantial rubidium-blockable potassium conductance located on the basal membrane of Locusta Malpighian tubule cells.

Pharmaceutical excipient development: the need for preclinical guidance.

Pharmaceutical excipients have a vital role in drug formulations, a role that has tended to be neglected as evidenced by the lack of mechanisms to assess excipient safety outside a new drug application process. Currently, it is assumed that an excipient is "approved" when the new drug formulation, of which it is a constituent, receives regulatory acceptance. Existing regulations and guidelines indicate that new (novel) excipients should be treated as new chemical entities with full toxicological evaluation. No guidance is available for potentially useful materials (essentially new excipients) available from other industries, e.g., food additives or for established excipients with a new application, e.g., dose route change. However, despite this situation, drug companies are actively evaluating new materials or applying new uses to established excipients. Recently developed excipients (e.g., materials giving "sugar-free" status to medical preparations, the cyclodextrins, and the hydrofluoroalkane inhalation propellants) and excipients undergoing development (e.g., chitosan, various enteric coating substances, liposomes, polymers derived from glycolic and lactic acids, and vaccine adjuvants) are all discussed. In light of many other areas of drug development having recently benefited from new or updated regulatory guidance, specific guidance to assist companies in the development of their excipients is urgently needed. Also, an excipient testing strategy would be an excellent topic for inclusion for International Conference on Harmonisation (ICH) consideration. Such guidance/discussion would complement the current advances in pharmacopoeial standardization of excipient quality. As a consequence, it may be possible to have excipients reviewed by a committee of an international pharmacopoeia with the safety data assessed by elected experts and published.

An assessment of two gastric transport models currently used in safety pharmacology testing.

1. The potential effects of new drugs on the digestive system can be examined in a number of model systems of which intestinal motility in the mouse and/or gastric emptying in the rat are examples recommended for safety pharmacology evaluation. 2. Intestinal motility, assessed by the transit of carmine dye in the mouse and gastric motility, assessed by stomach weight in the rat, were examined using a range of clinical drugs or potent pharmacological agents known to affect gastrointestinal function. Assessment of both models in the guinea-pig was also evaluated. 3. Activity was demonstrated with codeine, diazepam, atropine and CCK-8 (all of which inhibited gastric function). However, neither model gave consistent and reliable results with the remaining reference compounds, namely metoclopramide, bethanechol, cisapride, deoxycholate, carbachol and domperidone. 4. In conclusion, this investigation questions the usefulness of simple models of gastrointestinal transport in the rodent as a means of detecting potential effects of a new drug on the digestive system. This finding should be of concern to the pharmaceutical industry as these simple models are routinely used as part of a regulatory safety pharmacology 'package' of studies.

A toxicological review of lactose to support clinical administration by inhalation.

Although lactose is widely used in the pharmaceutical industry as an excipient in preparations given by several routes of administration, including by inhalation, there is no comprehensive review of its toxicological properties. This document seeks to review the available oral preclinical and clinical safety data from the literature, together with that generated by Fisons in animals using the inhalation route. In animal toxicity studies, lactose has been administered primarily by the inhalation and dietary routes to the rat, dog and/or primate. Adverse findings, such as abdominal distension and diarrhoea, have been demonstrated in rodent feeding studies. However, these changes are considered to be due to non-specific effects associated with high dietary doses of lactose, with a subsequent production of a dietary imbalance which results in physiological disturbances and an overload in the metabolic processes particularly involving calcium. These changes at high dietary intakes of lactose are considered to be of little relevance for man under the normal conditions of use of the material as an excipient in pharmaceutical formulations. No adverse local effects to the lung have been demonstrated in the animal studies using the inhalation route. Although the inhalation dose of lactose in the animal studies, of which most is subsequently swallowed, is markedly higher than the clinical dose, it is considerably less than consumed in animal studies using the dietary route. Consequently, it is not surprising that lactose is well tolerated by the inhalation route. In a small number of susceptible humans, intolerance to lactose is generally observed with oral intake of lactose, usually as a constituent of milk and is associated with lactase deficiency. Swallowed lactose at the levels present in inhaled preparations is unlikely to present any significant problems in patients with lactase deficiency. In conclusion, lactose is well recognized as a safe pharmaceutical excipient for use in oral or inhalation formulations and is not likely to constitute any significant toxicological hazard to man.