Monitoring of polycyclic aromatic hydrocarbons (PAH) in food supplements containing botanicals and other ingredients on the Dutch market.

Food supplements can contain polycyclic aromatic hydrocarbons (PAH). The European Food Safety Authority (EFSA) has defined 16 priority PAH that are both genotoxic and carcinogenic and identified eight priority PAH (PAH8) or four of these (PAH4) as good indicators of the toxicity and occurrence of PAH in food. The current study aimed to determine benzo[a]pyrene and other EFSA priority PAH in different categories of food supplements containing botanicals and other ingredients. From 2003 to 2008, benzo[a]pyrene exceeded the limit of quantification (LOQ) in 553 (44%) of 1258 supplements with a lower-bound mean of 3.37 µg kg(-1). In 2008 and 2009, benzo[a]pyrene and 12 other EFSA priority PAH were determined in 333 food supplements. Benzo[a]pyrene exceeded the LOQ in 210 (63%) food supplements with a lower-bound mean of 5.26 µg kg(-1). Lower-bound mean levels for PAH4 and PAH8(-indeno[1,2,3-cd]pyrene) were 33.5 and 40.5 µg kg(-1), respectively. Supplements containing resveratrol, Ginkgo biloba, St. John's wort and propolis showed relatively high PAH4 levels in 2008 and 2009. Before 2008, supplements with these ingredients and also dong quai, green tea or valerian contained relatively high benzo[a]pyrene levels. On average, PAH4 intake resulting from food supplement use will be at the lower end of the range of contributions of main food groups to PAH4 exposure, although individual food supplements can contribute significantly to PAH4 exposure. Regular control of EFSA indicator PAH levels in food supplements may prove a way forward to reduce further the intake of PAH from food.

Monitoring of mercury, arsenic, and lead in traditional Asian herbal preparations on the Dutch market and estimation of associated risks.

Traditional herbal preparations used in Ayurveda, traditional Chinese medicine, traditional Tibetan medicine, and other Asian traditional medicine systems may contain significant amounts of mercury, arsenic or lead. Though deliberately incorporated in Asian traditional herbal preparations for therapeutic purposes, these constituents have caused intoxications worldwide. The aim of this study was therefore to determine mercury, arsenic, and lead levels in Asian traditional herbal preparations on the Dutch market. A total of 292 traditional herbal preparations used in Ayurveda, traditional Chinese medicine, and traditional Tibetan medicine were sampled between 2004 and 2007. Samples were mostly multi-ingredient traditional herbal preparations containing herbs and minerals. The labeling of less than 20% of the traditional herbal preparations suggested the presence of mercury, arsenic or lead. These elements were shown by inductively coupled mass spectrometry (ICP-MS) in 186 (64%) of 292 traditional herbal preparations. Estimated weekly mercury, arsenic, and lead intake levels were calculated for each traditional herbal preparation from the analytically determined concentrations and the recommended dose. A total of 59 traditional herbal preparations (20%) were likely to result in intakes of these elements significantly exceeding safety limits. Of these 59 traditional herbal preparations, intake estimates for 50 traditional herbal preparations significantly exceeded the safety limit for mercury (range = 1.4-1747 mg week(-1)); intake estimates for 26 traditional herbal preparations significantly exceeded the safety limit for arsenic (range = 0.53-427 mg week(-1)) and intake estimates for eight traditional herbal preparations were significantly above the safety limit for lead (range = 2.6-192 mg week(-1)). It is concluded that the mercury, arsenic, and lead contents of traditional herbal preparations used in Ayurveda, traditional Chinese medicine, and traditional Tibetan medicine remain a cause for concern and require strict control.

Determination of the level of benzo[a]pyrene in fatty foods and food supplements.

A routine method was developed for the quantification of benzo[a]pyrene (BaP) in edible oils and food supplements. BaP is often taken as an indicator of the presence of polycyclic aromatic hydrocarbons. The method consists of on-line liquid chromatography clean-up followed by injection to an HPLC system with fluorescence detection. The method has good performance characteristics and gave good results in proficiency tests. From 2002 to 2004, about 1350 samples of oils and food supplements were analysed using this method to test the level of BaP. About 20% of the edible oils contained more than 1.2 microg kg-1 BaP (which is the limit applied by the Dutch Food and Consumer Product Safety Authority until 1 April 2005, and includes measurement uncertainty). In the case of food supplements, more then 30% contained too high levels of BaP, ranging from 1.2 to 135 microg kg-1.

Development of a rat model to assess the efficacy of the somatosensory-evoked potential as indicator of analgesia.

Drug-induced changes in somatosensory-evoked potentials (SEPs) are considered to reflect an altered nociceptive state. Therefore, the SEP is proposed to be a parameter of analgesic efficacy. However, at present, SEPs have not been studied in relation to animal pain. The present study aims to develop a rat model in which this relationship can be studied based on Pavlovian fear conditioning. Therefore, rats, implanted with epidural electro-encephalogram recording electrodes, were randomly assigned to either a paired or random-control group and subjected to an aversive-to-appetitive transfer paradigm. During the aversive phase, the SEP-stimulation paradigm (5 mA square wave pulses, n = 72, of 2 ms duration each, with a stimulus frequency of 0.5 Hz; total duration 144 s) was used as the unconditioned stimulus (US), while a tone (40 s, 1500 Hz, 85 dB sound pressure level) was used as the conditioned stimulus (CS). During the appetitive phase, the CS was presented paired to the presentation of a sugar pellet. When compared to the random-control group, the paired group showed significantly more freezing behavior and significantly less reward-directed behavior in response to the CS in the appetitive phase. In addition, SEPs were not significantly affected by fear conditioning. Based on these results, we conclude that the SEP-stimulation paradigm can be successfully employed as a US in fear conditioning. In future studies, fear conditioning can be carried out under different levels of an analgesic regimen to allow the changes in SEP parameters to be compared to changes in fear-induced behavior making this model potentially useful to validate SEP parameters as indicators of analgesia.

Middle-latency auditory-evoked potential in acepromazine-sedated dogs.

The middle-latency auditory-evoked potential (MLAEP) has been investigated as means of monitoring anesthesia in dogs. The goals of this study were to develop a technique to record MLAEPs in awake dogs and to determine the effects of sedation. The MLAEP was recorded in 12 dogs with and without sedation with acepromazine. Three needle electrodes were inserted SC. Click stimuli were delivered biaurally. Signal acquisition, averaging, and analysis were performed by software developed in-house. Signals were recorded for 128 milliseconds, and the responses to 1,024 stimuli were averaged. The waveforms from 10 recordings were averaged, and the amplitudes and latencies of peaks that could be consistently identified were measured. Data measured were compared by means of a paired 2-sided Student's t-test. Interpretable MLAEPs were recorded in 10 of the 12 dogs. Three peaks were consistently identified (Pa, Nb, and Pb). The latencies of these peaks were significantly (P = .032, .035, and .028, respectively) shorter in awake (mean +/- SD milliseconds) (Pa = 18.85 +/- 1.36, Nb = 30.50 +/- 3.55, and Pb = 47.70 +/- 5.53) than in sedated (Pa = 22.40 +/- 3.88, Nb = 35.75 +/- 6.77, and Pb = 55.30 +/- 10.55) dogs. The Pb amplitude was not significantly different (2.51 +/- 1.30 microV awake and 2.19 +/- 1.10 microV sedated). This study demonstrates that acepromazine sedation causes changes in MLAEP.

Click and low-, middle-, and high-frequency toneburst stimulation of the canine cochlea.

A method was developed to deliver tonebursts ranging in frequency from 1 to 32 kHz for frequency-specific assessment of the canine cochlea. Brainstem auditory-evoked responses (early latency responses, 0-10 ms) to a click (CS) and to 1-, 2-, 4-, 8-, 12-, 16-, 24-, and 32-kHz toneburst stimulations (TS) were compared at 80-dB sound pressure level stimulus (SPL) intensity in 10 adult dogs. All stimulations yielded a 5-7 positive wave pattern, with the exception of the 1-kHz TS, which evoked a frequency-following response (FFR). Thresholds were lowest for the CS and the 12- and 16-kHz TS. All individual peak latencies for TS were significantly (P < or = .05) longer than for CS. Peak I latencies were significantly (P < or = .05) shorter for the 12- and 16-kHz TS than for the other TS. Interpeak latencies I-V were significantly (P < or = .05) longer for the 4- to 32-kHz TS than for CS. Differences in interpeak latencies I-III were not significant. Amplitudes of waves I and V were significantly (P < or = .05) lower for TS than for CS, except for higher wave V amplitude (P < or = .05) at 2- and 32-kHz TS. Peak I-V amplitude ratios were significantly (P < or = .05) higher for the 2-, 4-, 16-, 24-, and 32-kHz TS and lower for the 8- and 12-kHz TS, compared to CS. We conclude that reproducible information on frequency specificity of the canine cochlea can be obtained by TS. This report provides a normative database for parameters needed to evaluate frequency-specific hearing loss in dogs.

The rat vertex-middle latency auditory-evoked potential as indicator of anaesthetic depth: a comparison with evoked-reflex testing.

We investigated whether components from the rat Vx-MLAEP could be used to assess depth of anaesthesia induced by propofol. Propofol decreased MLAEP amplitudes and increased latencies. We propose that the P(16)-N(22) wave in the rat MLAEP is similar to the human P1, and that recovery of this wave during propofol anaesthesia correlates with behavioural measures of the regaining of consciousness.

EEG evaluation of reflex testing as assessment of depth of pentobarbital anaesthesia in the rat.

Electroencephalography (EEG) was applied to evaluate the validity of the paw pinch reflex as an indicator of anaesthetic depth in rats which are anaesthetized with a single intraperitoneal dose of pentobarbital. After induction of the anaesthesia, characterized by the rapid loss of the animals' ability to maintain upright posture, the EEG of 10 out of 11 rats was dominated by paroxysmal (burst suppression) activity, associated with unconsciousness. In seven out of 11 rats, the paw pinch reflex was lost after onset of paroxysmal electroencephalographic activity. However, the paw pinch reflex remained present in four out of 11 animals, demonstrating that the response is independent of cortical activity. In five out of seven rats, the EEG still showed paroxysmal activity when the paw pinch reflex was regained. However, in two other rats the EEG returned to a pattern similar to that shown by awake animals, 4 and 21 min respectively, before the reflex was regained. These data indicate that in the pentobarbital-anaesthetized rat, presence of the paw pinch reflex is not related to the level of depression of electrical activity in the cerebral cortex, and consequently is probably not related to the level of consciousness. Based upon these findings it is concluded that the paw pinch reflex is unreliable as a sole indicator of anaesthetic depth.

[Euthanasia of piglets: gas or injection?]. / Euthanasie bij jonge biggen: gas of injectie?

Methods for the humane killing of animals are watched critically by both the public and the veterinary community. Evaluation of such methods requires assessment of efficacy as well as emotional and ethical aspects. Rapidity of loss of consciousness is a crucial factor in such evaluations. In the present study, four methods for piglet euthanasia were compared with regard to presence of indicators of discomfort (pain, anxiety, stress) and rapidity of onset of death, defined as the absence of breathing, heart beats and reflexes, combined with isoelectricity of the electro-encephalogram (EEG). The study was performed on piglets, which had to be destroyed on account of preventive measures against swine fever. The following methods were applied: CO2 98%, CO2/O2 65/35%, T61 and pentobarbital (Euthesate) injected intracardially. Intracardial injections of T61 and pentobarbital provide fast unconsciousness and death with minimal discomfort to the animal.