Considerations when deriving compound-specific limits for extractables and leachables from pharmaceutical products: Four case studies.

Leachables from pharmaceutical container closure systems are a subset of impurities that present in drug products and may pose a risk to patients or compromise product quality. Extractable studies can identify potential leachables, and extractables and leachables (E&Ls) should be evaluated during development of the impurity control strategy. Currently, there is a lack of specific regulatory guidance on how to risk assess E&Ls; this may lead to inconsistency across the industry. This manuscript is a cross-industry Extractables and Leachables Safety Information Exchange (ELSIE) consortium collaboration and follow-up to Broschard et al. (2016), which aims to provide further clarity and detail on the conduct of E&L risk assessments. Where sufficient data are available, a health-based exposure limit termed Permitted Daily Exposure (PDE) may be calculated and to exemplify this, case studies of four common E&Ls are described herein, namely bisphenol-A, butylated hydroxytoluene, Irgafos® 168, and Irganox® 1010. Relevant discussion points are further explored, including the value of extractable data, how to perform route-to-route extrapolations and considerations around degradation products. By presenting PDEs for common E&L substances, the aim is to encourage consistency and harmony in approaches for deriving compound-specific limits.

Chemical stabilization of polymers: Implications for dermal exposure to additives.

Technical benefits of additives in polymers stand in marked contrast to their associated health risks. Here, a multi-analyte method based on gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) was developed to quantify polymer additives in complex matrices such as low-density polyethylene (LDPE) and isolated human skin layers after dermal exposure ex vivo. That way both technical aspects and dermal exposure were investigated. The effects of polymer additivation on the material were studied using the example of LDPE. To this end, a tailor-made polymer was applied in aging studies that had been furnished with two different mixtures of phenol- and diarylamine-based antioxidants, plasticizers and processing aids. Upon accelerated thermo-oxidative aging of the material, the formation of LDPE degradation products was monitored with attenuated total reflectance-Fourier transformed infrared (ATR-FTIR) spectroscopy. Compared to pure LDPE, a protective effect of added antioxidants could be observed on the integrity of the polymer. Further, thermo-oxidative degradation of the additives and its kinetics were investigated using LDPE or squalane as matrix. The half-lives of additives in both matrices revealed significant differences between the tested additives as well as between LDPE and squalane. For instance, 2-tert-butyl-6-[(3-tert-butyl-2-hydroxy-5-methylphenyl)methyl]-4-methylphenol (Antioxidant 2246) showed a half-life 12 times lower when incorporated in LDPE as compared to squalane. As a model for dermal exposure of consumers, human skin was brought into contact with the tailor-made LDPE containing additives ex vivo in static Franz diffusion cells. The skin was then analyzed for additives and decomposition products. This study proved 10 polymer additives of diverse pysicochemical properties and functionalities to migrate out of the polymer and eventually overcome the intact human skin barrier during contact. Moreover, their individual distribution within distinct skin layers was demonstrated. This is exemplified by the penetration of the procarcinogenic antioxidant N-phenylnaphthalen-2-amine (Neozon D) into the viable epidermis and the permeation through the skin of the neurotoxic plasticizer N-butylbenzenesulfonamide (NBBS). In addition, the analyses of additive degradation products in the isolated skin layers revealed the presence of 2-tert-butyl-4-methylphenol in all layers after contact to a polymer with substances of origin like Antioxidant 2246. Thus, attention needs to be paid to absorption of polymer additives together with their degradation products when it comes to dermal exposure assessment.

Rational use of antioxidants in solid oral pharmaceutical preparations

Antioxidants are currently used as efficient excipients that delay or inhibit the oxidation process of molecules. Excipients are often associated with adverse reactions. Stability studies can guide the search for solutions that minimize or delay the processes of degradation. The ability to predict oxidation reactions in different drugs is important. Methods: This study was conducted to assess the rational use of butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), sodium metabisulfite (SMB), propyl gallate (PG) and cysteine (CYS) in tablet formulations of simvastatin and ketoconazole. These antioxidants were evaluated according to stability parameters and the relationship between efficiency of the antioxidant and chemical structure of the drugs. Results were compared with DPPH tests and computational simulations. BHT was most efficient regarding simvastatin stability, and the most effective BHT concentrations for maintaining stability were 0.5 and 0.1%. In relation to ketoconazole, SMB was most efficient for maintaining content and dissolution profile. The evaluation by DPPH showed that the largest percentage of absorbance reduction was observed for PG, while SMB proved most efficient and had lower consumption of DPPH. The same pattern was observed, albeit with lower efficiency, for the other lipophilic antioxidants such as BHT and BHA. The results of the molecular modeling study demonstrated that electronic properties obtained were correlated with antioxidant activity in solution, being useful for the rational development of liquid pharmaceutical formulations but not for solid oral formulations. This study demonstrated the importance of considering stability parameters and molecular modeling to elucidate the chemical phenomena involved in antioxidant activity, being useful for the rational use of antioxidants in the development of pharmaceutical formulations.

Atualmente, antioxidantes são usados como excipientes eficientes, que retardam ou inibem o processo de oxidação de moléculas. Excipientes são frequentemente associados a efeitos adversos. Estudos de estabilidade podem ajudar na busca por possíveis soluções para minimizar ou retardar os processos de degradação. A habilidade de prever as reações de oxidação em diferentes fármacos é importante. O estudo foi conduzido com o objetivo de avaliar o uso racional de hidroxianisol butilado (BHA), hidroxitolueno butilado (BHT), metabissulfito sódico (SMB), galato de propila (PG) e cisteína (CYS) em formulações de comprimidos de sinvastatina e cetoconazol. Eles foram avaliados por parâmetros de estabilidade e pela relação entre a eficiência dos antioxidantes e a estrutura química do fármaco. Os resultados foram comparados com testes de DPPH e simulações em computador. BHT foi mais eficiente com relação a estabilidade da sinvastatina e às concentrações mais eficientes para manutenção de estabilidade foram 0,5 e 0,1%. Com relação ao cetoconazol, SMB foi mais eficiente em manter o conteúdo e o perfil de dissolução. A avaliação por DPPH mostrou que o maior percentual de redução de absorção foi observado para PG, enquanto que SMB mostrou ser mais eficiente e consumir menos DPPH. A mesma tendência foi observada com menos eficiência em todos os outros antioxidantes lipofílicos como o BHT e BHA. Os resultados do estudo de modelagem molecular demonstraram que as propriedades eletrônicas obtidas podem ser correlacionadas com a atividade antioxidante em solução, sendo útil para o desenvolvimento racional de formulações farmacêuticas líquidas, mas não para formulações sólidas orais. Este estudo demonstrou a importância de considerar parâmetros de estabilidade e modelagem molecular para elucidar os fenômenos químicos envolvidos na atividade antioxidante, sendo úteis para o uso racional de antioxidantes no desenvolvimento de formulações farmacêuticas.

Metabolic activation of heterocyclic amines and expression of CYP1A1 in the tongue.

Xenobiotic metabolism in oral tissues, especially in the tongue, has never been reported. In the present study, the metabolic activation/detoxification ability of promutagens in the tongue and the expression levels of related enzymes were investigated. Quantitative PCR analysis of rat tongue demonstrated constitutive messenger RNA (mRNA) expression of numerous drug-metabolizing enzymes. In particular, we detected mRNA, protein expression, and enzymatic activity of cytochrome P450 (CYP)1A1 in the tongue tissue. Metabolic activation of promutagens in the tongue was estimated using benzo[a]pyrene or heterocyclic amines (HCAs), found in cooked meat and tobacco products. Metabolic activation levels of HCAs in the tongue were comparable to those in the liver. In contrast, the expression levels of glutathione-S-transferase (GST) and uridine diphosphate-glucuronosyltransferase (UGT) in the tongue were considerably lower compared with those in the liver, and as a result, the mutagenic activity in the tongue was not decreased by GST- or UGT-dependent conjugation. Treatment of rats with sudan III, a typical inducer of CYP1A1, resulted in markedly increased CYP1A1 mRNA, protein expressions, and CYP1A-dependent enzymatic and mutagenic activities. In addition, CYP1A1 mRNA expression in carcinoma cells (SAS) was induced by sudan III exposure. In conclusion, mutagenic activation of xenobiotics and an increased risk of cancer in the tongue were observed in this study. Furthermore, ingestion of drug-metabolizing enzyme inducers has the potential to increase the metabolic activation in the tongue tissue and increase the risk of biomolecular attack by promutagens.

The significance of excursions above the ADI: duration in relation to pivotal studies.

The significance of excursions of intake above the ADI, TDI, or PTWI can only be assessed by reference to the database which led to the derivation of these, most particularly the duration of the pivotal study (chronic, subchronic, acute), the pharmacokinetic parameters, and the nature of toxicity and mechanism of action. Although this implies a case by case assessment, a number of typical situations may be recognized: (1) The substance (usually a contaminant, not an additive) has a very long half-life leading to accumulation in target organs/tissues, e.g., Cd or dioxin. The chronic toxicity is manifested when critical concentrations are achieved in these tissues and there is a large difference between the acutely toxic dose and the chronic NOAEL. In such a case, the effect of excursions above the PTWI on tissue levels is readily calculated; peak excursions of several times the PTWI for short periods (days, weeks, or even months) or lower peak intakes for even longer periods (months to years) may be inconsequential provided that the integrated exposure over longer periods does not lead to critical steady-state tissue concentrations being achieved. (In such cases, it is clearly inappropriate to divide the PTWI by 7 and treat this as an ADI.) (2) A more common situation for food additives is where the ADI is based on a chronic study, but the t1/2 is short, i.e., the situation is one of chronic stress rather than cumulative toxicity. In such cases, e.g., BHA or BHT, the effects on the target organ (hyperplasia, foci of altered cells, etc.) can usually be identified in subchronic studies, although progressive changes may occur in chronic studies. Two subsituations then arise. First, when the effects seen at the LOAEL in subacute/subchronic studies are truly reversible (e.g., methemoglobinemia), short-term studies with a reversibility component may become pivotal in assessing the consequences of short-term excursions above the ADI. Second, when the short-term effects are not fully reversible, or are even progressive, the consequences of short-term peaks of intake above the ADI would require careful evaluation against the NOAEL or LOAEL in subacute or subchronic studies. (3) A rare situation might arise where the ADI is based on a chronic toxicity study but the margins between the chronic NOAEL and some aspects of acute toxicity may be small. For example, for a compound which behaved like retinol, the ADI might be based on chronic effects on the liver but at maternally nontoxic doses the substance may be teratogenic following acute exposure during early pregnancy. Clearly in such a situation, the acute NOAEL for teratogenicity would be used appropriately to evaluate the risks associated with short-term peaks of exposure, i.e., a different study may be pivotal in determining the effects of large excursions above the ADI than that which was used to calculate it. Clearly, these cases are not comprehensive but do provide a framework against which to discuss the potential effects of excursions above the ADI and to reach rational conclusions which are not based on the misapprehension that the ADI (or worse, the PTWI x 7) is a lower bound of toxicity.

Selective inhibition and induction of CYP activity discriminates between the isoforms responsible for the activation of butylated hydroxytoluene and naphthalene in mouse lung.

1. Selective induction and inhibition experiments have been used to identify the cytochrome P450 (CYP) isoforms responsible for butylated hydroxytoluene (BHT) bioactivation in mouse lung. 2. Pre-treatment of BALB/c mice with O,O,O-trimethylphosphorothioate (OOOMeP(S)), which prevented all the signs of toxicity observed following BHT treatment, inhibited the pulmonary activity of pentoxyresorufin O-dealkylase (PROD) and coumarin hydroxylase but not 4-nitrophenol hydroxylase. 3. Pulmonary coumarin hydroxylase activity was greater in DBA than in BALB/c mice but the severity of BHT-induced lung injury was similar. 4. Pre-treatment with pyrazole, which exacerbated BHT-induced lung injury, did not affect pulmonary coumarin hydroxylase or 4-nitrophenol hydroxylase activity but increased that of PROD. 5. Pre-treatment with OOOMeP(S) prevented the lethargy and weight-loss associated with naphthalene poisoning but not the pulmonary injury. Pre-treatment with pyrazole did not exacerbate naphthalene-induced injury. 6. Members of both CYP2F and 2B sub-families have been shown to exhibit PROD activity and 2F2 activates naphthalene in mouse lung. The current studies, however, indicate that 2F2 is unlikely to be a significant component of PROD activity in mouse lung. 2F2, like coumarin hydroxylase (2A5) and 4-nitrophenol hydroxylase (2E1), is not responsible for the pulmonary activation of BHT, which is largely attributable to an isoform of 2B, probably 2B10.

Tissue distribution of LY231617, an antioxidant with neuroprotectant activity, in the rat.

The tissue distribution of a butylated phenol antioxidant, LY231617, which has been shown to exert potent neuroprotection action was examined. Preliminary pharmacokinetic examination suggests that LY231617 was eliminated in a biphasic fashion after iv administration to the rat with a distribution half-life of 5 min and an elimination half-life of close to 2.5 h. The volume of distribution of the compound was large (7.4 L), consistent with its lipophilic structure. Dosing paradigms that have historically resulted in pharmacologically relevant activity were examined and were found to generate brain tissue levels of LY231617 of approximately 45 micrograms/g.

Metabolic activation of butylated hydroxytoluene by mouse bronchiolar Clara cells.

Metabolism of BHT (2,6-di-tert-butyl-4-methylphenol) is requisite for its pneumotoxic activities. Previous evidence using microsomal preparations from livers and lungs of mice indicated that cytochrome P450-catalyzed hydroxylation of a tertbutyl group to form 6-tert-butyl-2-(hydroxy-tert-butyl)-4-methylphenol (BHTOH) is the first step in the bioactivation of this compound. Subsequent oxidation of BHTOH produces the quinone methide 6-tert-butyl-2-(hydroxy-tert-butyl)-4-methylene-2,5-cyclohexadienone (BHTOH-QM), and this highly reactive electrophile may be directly responsible for the pulmonary effects of BHT. The present study assessed the ability of intact bronchiolar Clara cells isolated from mice, a major site of pulmonary xenobiotic metabolism, to convert BHT to BHTOH-QM. The data demonstrate that BHTOH is, in fact, the principal oxidation product in these cells, and that a substantial portion of this metabolite is oxidized further to the quinone methide. BHTOH was found to be considerably more toxic to Clara cells than BHT, and both toxicity and metabolism were simultaneously depressed by the cytochrome P450 inhibitor SKF 525-A. These results strongly support the hypothesis that BHTOH-QM is the active metabolite that generates acute pneumotoxicity and modulates lung tumor formation.

Cytochrome P450 2B isoenzymes are responsible for the pulmonary bioactivation and toxicity of butylated hydroxytoluene, O,O,S-trimethylphosphorothioate and methylcyclopentadienyl manganese tricarbonyl.

O,O,S-Trimethylphosphorothioate and methylcyclopentadienyl manganese tricarbonyl damage the type I pneumocytes of the alveolar epithelium in rats. Butylated hydroxytoluene causes similar damage in mice. The toxicity of these compounds is dependent on their bioactivation by the cytochrome P-450 (CYP) system. A range of compounds, that modifies the activity of specific CYP isoenzymes, has been used to establish those particular isoenzymes involved in bioactivation. Pulmonary toxicity was assessed by measurement of lung weight and changes in the activity of gamma-glutamyltranspeptidase and alkaline phosphatase in bronchoalveolar lavage fluid. O,O,S-Trimethylphosphorodithioate, bromophos, p-xylene and 2,4-dichloro-(6-phenyl-phenoxy)ethylamine all inhibited the dealkylation of pentoxyresorufin, an indicator of CYP2B1 activity, and also prevented pulmonary toxicity. There was a significant negative correlation between the level of pulmonary pentoxyresorufin dealkylation after pretreatment with O,O,S-trimethylphosphorodithioate and the severity of lung injury. This pretreatment also reduced the toxicity of butylated hydroxytoluene by a factor of 20 and methylcyclopentadienyl manganese tricarbonyl by a factor of 10. Modification of the activity of CYP1A1, CYP2E1 and CYP4B1 did not alter the toxicity of these compounds. These results indicate that pulmonary CYP2B1 is responsible for the bioactivation and toxicity of O,O,S-trimethylphosphorothioate and methylcyclopentadienyl manganese tricarbonyl in rats and the orthologous 2B isoenzyme in mice activates butylated hydroxytoluene.

Differential effects of butylated hydroxytoluene analogs on bull sperm subjected to cold-induced membrane stress.

Previous reports established that butylated hydroxy toluene (BHT) minimized cold-induced membrane rupture in sperm from several species. No data regarding the specificity of its effect is available. In this study 25 BHT analogs were tested for their effect on bovine sperm membrane stability. Fourteen were membrane lytic at 25 degrees C and 6 were neither membrane lytic nor membrane stabilizing. The remaining 5 compounds, a family of 2,6-tert-butyl phenols with substitutions at position 4 of hydrogen, methyl (BHT), ethyl, butyl, hexyl, or octyl, afforded effective membrane protection to cold shock. Since membrane protection is a function of both the ability of a compound to partition into the membrane and a molecule's effectiveness once there, an analysis of each analog's membrane partitioning, assessed by measuring the cellular analog/cholesterol ratio, showed the following extents of transfer for the analogs: ethyl = butyl greater than methyl = hydrogen greater than hexyl greater than octyl. Thus, an optimum chain length exists for partitioning from micellar donors into cells. A separate experiment established that all analogs, when incorporated in equivalent amounts, protect equally plasma and mitochondrial membranes from cold shock. No effect on acrosomal membrane stability was noted. BHT, but not the other analogs, reduced sperm motility. Addition of egg yolk to extender containing BHT analog protected sperm motility from cold shock but had little effect on membrane stabilization. Analysis of sperm membrane compartments revealed that little to no analog was partitioned into the outer acrosomal membrane or the plasma membrane overlying the acrosome, but rather was localized in other portions of the sperm. We conclude that (a) the effective BHT analogs, if partitioned into the membrane, are indistinguishable with regard to their capacity to eliminate cold-induced membrane lysis; (b) membrane-linked events (e.g., motility) are uniquely disrupted by a subset of this analog family; and (c) when concentrations of egg yolk and BHT analogs are carefully controlled, unique synergistic effects are noted.

Gastric retention and delayed absorption of a large dose of butylated hydroxytoluene in the rat.

1. After a single oral dose of 800 mg/kg of butylated hydroxytoluene to rats, the plasma concentration of 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (BHT quinone methide), an active metabolite of BHT, reached a maximum 18 h after dosing. 2. The gastrointestinal content of BHT remained constant from 0.5 to 12 h, and began to decline 18 h after dosing. 3. BHT concentrations in epididymal and subcutaneous adipose tissues also attained maxima 18 h after dosing. 4. Volumes and weights of stomachs and contents, and volumes of gastric contents of rats given 800 mg/kg BHT, were 2-3 times larger than those of controls 4-7 h after dosing while there were no significant differences at 24-27 h after dosing. 5. Retention of ingested material in the stomachs was also observed when 18-h starved rats were given 800 mg/kg BHT. 6. Thirty minutes after intraduodenal administration of 800 mg/kg BHT to anaesthetized rats. BHT, but not BHT quinone methide, was 0.4-1.4 micrograms/ml in portal vein plasma. No BHT was detected in plasma from the aorta descendens. BHT and/or BHT radical, and BHT alone, were also found in liver and epididymal adipose tissue at 7-20 and 25-40 micrograms/g wet weight, respectively. 7. These results indicate that delay in attainment of maximum concentration of BHT or its metabolites in internal organs may be due to the long retention in the stomach, caused by high dosage and the inhibitory effect of BHT on gastric function. Moreover, most BHT may not enter lymphatic fluid but it can be absorbed into portal blood.

Effect of route of administration on the relative efficacy and potency of 2-aminoethyl-4-(1,1-dimethylethyl)-6-iodophenol HCl (MK447) and furosemide.

2-Aminoethyl-4-(1,1-dimethylethyl)-6-iodophenol hydrochloride (MK447) is a chemically novel diuretic that has oral efficacy and potency greater than furosemide (FUR). In this study, the relative efficacy and potency of MK447 and FUR were assessed following oral, i.v. bolus and i.v. infusion administration in rats and dogs. MK447 was more potent than furosemide but the two were equally efficacious when administered orally to rats and dogs. The relative potency remained unchanged when administered as an i.v. bolus to rats (1 mg/kg). However, when infused continuously there was no difference in potency or efficacy between the two compounds. These observations lead to the conclusion that pharmacokinetic differences between the two compounds play a major role in determining their relative oral potency. Comparative studies of the effects of compounds given by different routes of administration are useful in providing insight into the role of pharmacokinetics in overall response to different compounds.

Formation and reactivity of alternative quinone methides from butylated hydroxytoluene: possible explanation for species-specific pneumotoxicity.

Previous work has shown that butylated hydroxytoluene [2,6-di-tert-butyl-4-methylphenol (BHT)] undergoes pi-oxidation in liver microsomes to form the quinone methide 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (QM). This electrophilic species binds covalently to glutathione and protein thiols and is believed to initiate pulmonary toxicity in mice. In the present investigation, we identified another quinone methide metabolite of BHT, 6-tert-butyl-2-(hydroxy-tert-butyl)-4-methylene-2,5-cyclohexadienone (QM-OH), formed subsequent to the microsomal hydroxylation of BHT at a tert-butyl group. Mouse liver and lung microsomes generate the two quinone methides, and evidence was obtained that both metabolites also are formed in vivo. In contrast, rat microsomes produce QM almost exclusively, with only traces of QM-OH formed in liver and none in lung. Studies of the chemical reactivities of the two quinone methides with GSH demonstrated that QM-OH reacts about 6-fold faster than QM. Infrared spectra, 1H NMR spectra, and electrochemical measurements all support the proposal that the enhanced electrophilicity of QM-OH is due to intramolecular hydrogen bonding of the ring oxygen with the side-chain hydroxyl. The results provide evidence, therefore, that the previous metabolic scheme for bioactivation of BHT to a pulmonary toxin should be amended to include tert-butyl hydroxylation and subsequent pi-oxidation to the activated electrophile QM-OH. This scheme is consistent with published data concerning BHT-induced pulmonary toxicity and provides an explanation for the species specificity of this effect.

Disposition of single oral doses of butylated hydroxytoluene in man and rat.

The kinetics and metabolism of butylated hydroxytoluene (BHT) in man and rats have been compared. Single oral doses of 200, 63 or 20 mg BHT/kg body weight were administered to rats and a single oral dose of 0.5 mg/kg body weight was ingested by human volunteers (non-smoking males). In rats, kinetic parameters (area under the plasma concentration-time curve, plasma BHT peak levels) showed a dose-dependent increase. Plasma BHT levels after oral administration were about four times higher than those that have been reported for another synthetic food antioxidant, butylated hydroxyanisole (BHA; Verhagen et al., Fd Chem. Toxic. 27, 151-158). This may be a reflection of a smaller volume of distribution for BHT, since there were no differences in plasma elimination half-life or plasma clearance between BHT and BHA. In man, the mean plasma concentration-time profile after oral BHT intake was well below the BHT profiles observed for rats and closely followed plasma BHA kinetics in man. In rats, the simultaneous administration of BHT (200 mg/kg body weight) and BHA (200 mg/kg) significantly decreased the absorption of BHT from the gastro-intestinal tract in the first few hours after treatment; the plasma kinetics of BHA were not influenced by the simultaneous administration of BHT. In human female volunteers no alterations in plasma BHT or BHA profiles were seen after the simultaneous ingestion of BHT (0.25 mg/kg body weight) and BHA (0.25 mg/kg). Rats excrete about 10% of an oral dose of 200 mg BHT/kg as unchanged BHT in the faeces, whereas in man no BHT could be detected in the faeces. Urinary excretion of (un)conjugated 3,5-di-tert-butyl-4-hydroxybenzoic acid (BHT-COOH) accounts for only a small percentage of the administered dose in both rats and humans. It is concluded that the plasma BHT concentrations reached after the administration of a single medium to high dose of BHT to rats or a single low dose to man are very different.

Short-term effects of butylated hydroxytoluene on the Wistar rat liver, urinary bladder and thyroid gland.

Long-term feeding of butylated hydroxytoluene (BHT) to rats and mice has been linked to the enhancement of the incidence of liver tumors. It is shown in this paper that in the liver, urinary bladder and thyroid of the male Wistar rat, feeding the highest tolerated doses of BHT for 30 days does not lead to detectable increases in [3H]thymidine labeling. On the other hand, treatment of rats with 0.5% dietary BHT leads to a time-limited increase in liver cell [3H]thymidine labeling that subsided to control values within 8 days. This increase in [3H]thymidine labeling in the liver is accompanied by an unexpectedly large increase in the mitotic index. These results are discussed in the light of the behavior of certain rodent liver tumorigens.

Extent of cutaneous metabolism during percutaneous absorption of xenobiotics.

In vitro percutaneous absorption studies generally do not determine whether biotransformation occurs during passage of a substance through the skin. Since it has recently been demonstrated that several chemicals are metabolized during skin permeation, we investigated the metabolism of five additional compounds (14C-labeled) after application to fuzzy rat skin: caffeine, p,p'-DDT, butylated hydroxytoluene (BHT), salicylic acid, and acetyl ethyl tetramethyltetralin (AETT). The viability of skin was maintained with a tissue culture medium. Radioactivity of each substrate and any metabolites in skin and receptor fluid was measured so that the absorption and metabolism of water-insoluble compounds would be accurately determined. Percutaneous absorption ranged from a low of 13% of the applied dose for BHT to a high of 49% for DDT. BHT was metabolized in skin to 4-hydroxy-BHT and an unknown metabolite. Of the absorbed radioisotope, 6.6% was isolated in biotransformed products found mainly in the receptor fluid. AETT was also metabolized during absorption, with 1.9% of the absorbed radioisotope found in two unknown peaks. Caffeine, DDT, and salicylic acid were not metabolized during skin permeation. Skin and liver microsomal metabolism was measured for all compounds except DDT. Metabolism in skin was observed only for the compounds also biotransformed in the diffusion cell; BHT and AETT were metabolized at 113 and 2.5 pmol/min/mg protein, respectively. In this study, as in others, skin metabolism was substantially less than the corresponding metabolism in liver. Therefore, a low rate of liver metabolism such as that found for caffeine, salicylic acid, and DDT might often be predictive of the absence of measurable metabolism during skin permeation. It seems likely that for many compounds, the biotransformations in skin will be small in terms of the percentage of absorbed material that is metabolized. Nevertheless, with potent compounds, even small quantities of a metabolite can be important and for pharmacokinetic studies, viability of skin must be maintained.

[Antiulcer activity of dibunol in experimental stomach and duodenal ulcers]. / Protivoiazvennaia aktivnost' dibunola pri éksperimental'nykh iazvakh zheludka i dvenadtsatiperstnoi kishki.

The antiulcer effect of two medicinal forms (oil solution, liniment) of dibunol in rats with different models of the stomach and duodenum ulcers was studied. The therapeutic activity of dibunol was evaluated by determining the index of ulcer formation as well as during the morphological and histochemical studies. The two medicinal forms of dibunol possessed equal antiulcer activity by all studied criteria. One should note a characteristic property of dibunol--its ability to produce pronounced epithelialization of ulceroerosive and ulceronecrotic lesions of the mucosa without rough scarring.