Assessing safety of extractables from materials and leachables in pharmaceuticals and biologics - Current challenges and approaches.

Leachables from pharmaceutical container closure systems can present potential safety risks to patients. Extractables studies may be performed as a risk mitigation activity to identify potential leachables for dosage forms with a high degree of concern associated with the route of administration. To address safety concerns, approaches to toxicological safety evaluation of extractables and leachables have been developed and applied by pharmaceutical and biologics manufacturers. Details of these approaches may differ depending on the nature of the final drug product. These may include application, the formulation, route of administration and length of use. Current regulatory guidelines and industry standards provide general guidance on compound specific safety assessments but do not provide a comprehensive approach to safety evaluations of leachables and/or extractables. This paper provides a perspective on approaches to safety evaluations by reviewing and applying general concepts and integrating key steps in the toxicological evaluation of individual extractables or leachables. These include application of structure activity relationship studies, development of permitted daily exposure (PDE) values, and use of safety threshold concepts. Case studies are provided. The concepts presented seek to encourage discussion in the scientific community, and are not intended to represent a final opinion or "guidelines."

Lack of micronuclei formation in bone marrow of rats after repeated oral exposure to nickel sulfate hexahydrate.

Workplace exposures to mixtures of nickel compounds have been associated with excess respiratory cancer risk. Animal studies with individual nickel compounds indicate that not all nickel substances have the same potency or potential to induce tumors. The bioavailability of nickel ions at critical cellular sites seems to be important to determine the potential of a substance to induce tumors in animals, but much less is understood about the exact nature (genotoxic or non-genotoxic) of the nickel effects. Within many regulatory frameworks (e.g., European Union), substances are classified for mutagenicity based on the available data and this classification will often influence the mode of action assigned to carcinogenic substances and the way in which risk assessment will be conducted. The objective of this study was to evaluate the ability of nickel sulfate hexahydrate to induce micronuclei in polychromatic erythrocytes (PCEs) in rat bone marrow. This study was conducted according to OECD and EU protocol guidelines. In the dose range-finding assays, the maximum tolerated dose was estimated to be 500 mg/kg/day. The doses used in the micronucleus assay were 125, 250, and 500 mg/kg/day. At least 2000 PCEs per animal were analyzed for micronuclei in PCEs. Cytotoxicity was assessed by scoring a minimum of 500 consecutive total polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). Nickel sulfate hexahydrate did not induce statistically significant increases in micronucleated PCEs at any dose examined. The negative results in the present study contribute significantly to the weight of evidence evaluation of the mutagenicity (chromosomal level) of nickel substances. These results are consistent with a non-genotoxic mode of action for soluble nickel that could explain the enhancement of cancer risk seen among refinery workers with mixed exposures and its lack of carcinogenicity in animal studies with single exposures.

Genotoxicity studies with pure trans-capsaicin.

Both positive and negative effects have been found in classical genetic toxicology assays with capsaicin. However, the capsaicin tested in most studies has been derived from pepper plant extracts, which is likely to display varying degrees of purity and possibly diverse impurity profiles. Therefore, the objective of the series of studies reported here was to test the genotoxic potential of pure, synthetic trans-capsaicin (the only naturally occurring geometric isomer of capsaicin), using four genotoxicity assays widely used to evaluate drug substances. These included the Ames, mouse lymphoma cell mutation, mouse in vivo bone marrow micronucleus and chromosomal aberration in human peripheral blood lymphocytes (HPBL) assays. In the Ames assay, pure trans-capsaicin was not mutagenic to Salmonella typhimurium or Escherichia coli when dissolved in dimethylsulfoxide and tested at concentrations extending into the toxic range. trans-Capsaicin was weakly mutagenic in mouse lymphoma L5178Y cells, in the presence of S9 mix, when dissolved in dimethylsulfoxide and tested at concentrations extending into the toxic range. Limited evidence for very weak activity was also obtained in the absence of S9 mix. trans-Capsaicin did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 800 mg/kg per day in male and 200 mg/kg per day in female CD-1 mice using a 0 h plus 24 h oral dosing and 48 h sampling regimen. Finally, trans-capsaicin did not induce structural or numerical chromosomal aberrations when evaluated for its ability to induce clastogenicity in blood lymphocytes. Taken together, these data suggest that the genotoxic potential of pure trans-capsaicin is very low, especially as the clinical significance of weak mutagenicity in the mouse lymphoma assay for catechol-moiety containing compounds is unclear. Moreover, the different genotoxicity profiles of pure trans-capsaicin and purified chili pepper extracts suggest that the purity and source of capsaicin should always be an important consideration for toxicological evaluations.