Hirmi Valley liver disease: a disease associated with exposure to pyrrolizidine alkaloids and DDT.

BACKGROUND & AIMS: Hirmi Valley liver disease was first reported in 2001 in Tigray, Ethiopia. 591 cases, including 228 deaths, were reported up to December 2009. The pyrrolizidine alkaloid acetyllycopsamine was detected in stored grain and residents reported adding the pesticide DDT (dichlorodiphenyldichloroethylene) directly to their food stores. We aimed to characterise the clinical features of the disease, and explore the role of these chemicals in its aetiology. METHODS: 32 cases were examined and full clinical histories taken. Nine cases underwent liver biopsy in hospitals. Serum and urine samples were collected from cases and controls. Urine was analysed for acetyllycopsamine by UPLC-MS. Total DDT in serum was measured by ELISA. Hepatotoxicity of DDT and acetyllycopsamine alone or in combination was explored in C57BL/6J mice. RESULTS: Clinical presentation included epigastric pain, abdominal swelling, bloody diarrhoea, hepatomegaly, splenomegaly, and ascites. Histology revealed acute injury characterised by centrilobular necrosis or chronic injury with bile ductular reaction, cytomegaly and fibrosis but no hepatic vein occlusion. Acetyllycopsamine was detected in urine samples taken in the affected area with significantly greater concentrations in 45 cases than in 43 controls (p=0.02). High levels of DDT (>125 ppb) were detected in 78% of serum samples. In mice, DDT (3 × 75 mg/kg) significantly increased the hepatotoxicity (plasma ALT, p=0.0065) of acetyllycopsamine (750 mg/kg), and in combination induced liver pathology similar to Hirmi Valley liver disease including centrilobular necrosis and cytomegaly. CONCLUSIONS: This novel form of disease appears to be caused by co-exposure to acetyllycopsamine and DDT.

Ozone Generation during High-Voltage Leak Detection: Fiction or Reality?

In order to further clarify if and how much ozone is generated during high-voltage leak detection and to identify measures to reduce the impact of ozone generation on product quality, a highly sensitive analytical system was employed to investigate the generation of ozone at different operational conditions of high-voltage leak detection integrity testing. The analytical system is based on oxidation of Iodide ions in solution and identification of the Iodine formed by N, N-Diethyl-p-phenylendiamine (DPD) according to DIN 38403. Sensitivity of the system was found suitable to detect ozone levels as low as 0.025 ppm (mg/L). High-voltage leak detection process parameters-inspection speed, high voltage, filling level of the ampoule, and exposure time to the ampoule to high voltage-were varied between maximum and minimum values applicable in integrity testing of different ampoule sizes. For variation of exposure time, ampoules were repetitively tested by the leak testing machine to achieve a maximum exposure time of the ampoule up to 24 s (exposure time during production ≤2.4 s). No ozone was detected during the study under all inspection conditions. Even repeated exposure of the ampoules to high-voltage leak detection did not result in generation of measurable ozone levels. It has to be concluded that high voltage leak detection is not prone to causing oxidation of the drug products.LAY ABSTRACT: For drug products in ampoules, 100% integrity testing is requested both by European and US regulations. Detection of integrity defects like small holes or cracks in the ampoule can be performed by numerous methods. Due to automation requirements, only three methods have been used during routine production-vacuum decay, head space infrared spectroscopy, and high-voltage leak detection. High-voltage leak detection is the most common method used in the pharmaceutical industry for 100% integrity testing of ampoules, but resent publications showed considerable oxidation of an air-sensitive active pharmaceutical ingredient during the integrity testing process. It was assumed that ozone generated in the inside of the vial due to exposure of the product to the high voltage during the testing process may be responsible for this result. As ozone is a very aggressive and non-selective oxidant, this result posed a serious thread not only to this product but also to the quality of many other products subjected to integrity testing by high-voltage leak detection.

High-Voltage Leak Detection of a Parenteral Proteinaceous Solution Product Packaged in Form-Fill-Seal Plastic Laminate Bags. Part 3. Chemical Stability and Visual Appearance of a Protein-Based Aqueous Solution for Injection as a Function of HVLD Exposure.

This Part 3 of this three-part research series reports the impact of high-voltage leak detection (HVLD) exposure on the physico-chemical stability of the packaged product. The product, intended for human administration by injection, is an aqueous solution formulation of the rapid acting insulin analogue, insulin aspart (NovoRapid®/NovoLog®) by Novo Nordisk A/S, Bagsværd, Denmark. The package is a small-volume form-fill-seal plastic laminate bag. Product-packages exposed to HVLD were compared to unexposed product after storage for 9 months at recommended storage conditions of 5 ± 3 °C. No differences in active ingredient or degradation products assays were noted. No changes in any other stability indicating parameter results were observed. This report concludes this three-part series. Part 1 documented HVLD method development and validation work. Part 2 explored the impact of various package material, package temperature, and package storage conditions on HVLD test results. Detection of leaks in the bag seal area was investigated. In conclusion, HVLD is reported to be a validatable leak test method suitable for rapid, nondestructive container-closure integrity evaluation of the subject product-package. LAY ABSTRACT: In Part 1 of this three-part series, a leak test method based on electrical conductivity and capacitance, also called high-voltage leak detection (HVLD), was proven to find hole leaks in small plastic bags filled with a solution of insulin aspart intended for human injection (NovoRapid®/NovoLog® by Novo Nordisk A/S, Bagsværd, Denmark). In Part 2, the ability of the HVLD method to find other types of package leaks was tested, and the impact of package material and product storage temperature on HVLD results was explored. This final Part 3 checked how well the packaged protein drug solution maintained its potency after HVLD exposure over 9 months of storage under long-term stability conditions. Results showed that HVLD caused no harm to the product.

High-voltage leak detection of a parenteral proteinaceous solution product packaged in form-fill-seal plastic laminate bags. Part 2. Method performance as a function of heat seal defects, product-package refrigeration, and package plastic laminate lot.

Part 1 of this three-part research series detailed the development and validation of a high-voltage leak detection test (HVLD, also known as an electrical conductivity and capacitance test) for verifying the container-closure integrity of a small-volume laminate plastic bag containing an aqueous solution formulation of the rapid-acting insulin analogue, insulin aspart (NovoRapid®/NovoLog®) by Novo Nordisk A/S, Bagsværd, Denmark. Leak detection capability was verified using positive controls each with a single laser-drilled hole in the bag film face. In this Part 2, HVLD leak detection capability was further explored in four separate studies. Study 1 investigated the ability of HVLD to detect weaknesses and/or gaps in the bag heat seal. Study 2 checked the HVLD detection of bag holes in packages stored 4 days at ambient conditions followed by 17 days at refrigeration. Study 3 examined HVLD test results for packages tested when cold. Study 4 compared HVLD test results as a function of bag plastic film lots. The final Part 3 of this series will report the impact of HVLD exposure on product visual appearance and chemical stability. LAY ABSTRACT: In Part 1 of this three-part series, a leak test method based on electrical conductivity and capacitance, also called high-voltage leak detection (HVLD), was used to find leaks in small plastic bags filled with a solution for injection of the rapid-acting insulin analogue, insulin aspart (NovoRapid®/NovoLog®) by Novo Nordisk A/S, Bagsværd, Denmark. In this Part 2, HVLD leak detection capability was further explored in four separate studies. Study 1 investigated the ability of HVLD to detect bag heat seal leaks. Study 2 checked HVLD's ability to detect bag holes after a total of 21 days at ambient plus refrigerated temperatures. Study 3 looked to see if HVLD results changed for packages tested when still cold. Study 4 compared HVLD results for multiple bag plastic film lots. The final Part 3 of this series will report any evidence of drug component degradation caused by HVLD exposure.

High-voltage leak detection of a parenteral proteinaceous solution product packaged in form-fill-seal plastic laminate bags. Part 1. Method development and validation.

In Part 1 of this three-part research series, a leak test performed using high-voltage leak detection (HVLD) technology, also referred to as an electrical conductivity and capacitance leak test, was developed and validated for container-closure integrity verification of a small-volume laminate plastic bag containing an aqueous solution for injection. The sterile parenteral product is the rapid-acting insulin analogue, insulin aspart (NovoRapid®/NovoLog®, by Novo Nordisk A/S, Bagsværd, Denmark). The aseptically filled and sealed package is designed to preserve product sterility through expiry. Method development and validation work incorporated positive control packages with a single hole laser-drilled through the laminate film of each bag. A unique HVLD method characterized by specific high-voltage and potentiometer set points was established for testing bags positioned in each of three possible orientations as they are conveyed through the instrument's test zone in each of two possible directions-resulting in a total of six different test method options. Validation study results successfully demonstrated the ability of all six methods to accurately and reliably detect those packages with laser-drilled holes from 2.5-11.2 µm in nominal diameter. Part 2 of this series will further explore HVLD test results as a function of package seal and product storage variables. The final Part 3 will report the impact of HVLD exposure on product physico-chemical stability. LAY ABSTRACT: In this Part 1 of a three-part research series, a leak test method based on electrical conductivity and capacitance, called high voltage leak detection (HVLD), was used to find leaks in small plastic bags filled with an insulin pharmaceutical solution for human injection by Novo Nordisk A/S (Bagsværd, Denmark). To perform the test, the package is electrically grounded while being conveyed past an electrode linked to a high-voltage, low-amperage transformer. The instrument measures the current that passes from the transformer to the electrode, through the packaged product and along the package walls, to the ground. Plastic packages without defect are relatively nonconductive and yield a low voltage reading; a leaking package with electrically conductive solution located in or near the leak triggers a spike in voltage reading. Test methods were optimized and validated, enabling the detection of leaking packages with holes as small as 2.5 µm in diameter. Part 2 of this series will further explore HVLD test results as a function of package seal and product storage variables. The final Part 3 will report the impact of HVLD exposure on product stability.