Exposure data for cosmetic products: lipstick, body lotion, and face cream.

Accurate exposure information for cosmetic products and ingredients is needed in order to conduct safety assessments. Essential information includes both the amount of cosmetic product applied, and the frequency of use. To obtain current data, a study to assess consumer use practices was undertaken. The study included three widely used cosmetic product types: lipstick, body lotion, and face cream. Three hundred and sixty women, ages 19-65 years, who regularly use the products of interest, were recruited at ten different geographical locations within the US. The number of recruits was chosen to ensure a minimum of 300 completes per product type. Subjects were provided with prototype test products, and kept diaries and recorded detailed daily usage information over a two week period. Products were weighed at the start and completion of the study in order to determine the total amount of product used. Statistical analysis of the data was conducted to derive summary distribution of use patterns. The mean and median usage per application, respectively, for the three products was: face cream, 1.22 g and 0.84 g; lipstick, 10 mg and 5 mg; and body lotion, 4.42 g and 3.45 g. The mean and median usage per day for the three products was: face cream, 2.05 g and 1.53 g; lipstick, 24 mg and 13 mg; and body lotion, 8.70 g and 7.63 g. The mean number of applications per day for face cream and lipstick was 1.77 and 2.35, respectively. For body lotion, the mean number of applications per day was dependent on body area, and was 2.12, 1.52, 1.11, 0.95, 0.43, 0.26, and 0.40 for hands, arms, legs, feet, neck and throat, back, and other body areas, respectively. The effect of product preference on use practices was also investigated. This study provides current cosmetic exposure information for commonly used products which will be useful for risk assessment purposes.

Comparing and evaluating alternative (in vitro) tests on their ability to predict the Draize maximum average score.

The Cosmetic, Toiletry, and Fragrance Association (CTFA) Evaluation of Alternatives Program comprised a multi-phased study of the relationship between Draize eye irritation test data and comparable data from a selection of promising alternative (in vitro) tests. The CTFA Program was designed to determine the effectiveness and limitations of several in vitro tests over a range of different cosmetic and personal-care product types. Test materials constituted experimental formulations representative of three distinct product types. Each material was tested in vivo (according to a modified Draize eye irritation test protocol) and in vitro (according to one of up to forty different protocols). A statistical ranking and selection procedure ("concordance analysis") was used to identify those in vitro tests where the relationships between in vitro and in vivo score was sufficiently well defined to warrant further statistical analysis. In vitro test performance was then evaluated by regression modelling of these relationships. Maximum average Draize score (MAS) was utilized as the primary quantitative measure of eye irritation potential in vivo. The goodness-of-fit of the observed data to the regression model and comparison of the magnitude of upper and lower prediction-bounds on the range of probable MAS values associated with the regression model fit (prediction intervals) provide a means by which the performance of each in vitro test may be measured relative to Draize test outcome. The narrower the prediction interval (i.e. the more precise the fit), the more predictive of in vivo score (MAS) is the in vitro test result. The prediction interval thus represents uncertainty associated with Draize test prediction. Such uncertainty depends heavily on the degree of irritancy. In Phases I and II, the widths of the prediction intervals were narrowest in the region corresponding to low irritation potential; increasing widths were observed as irritation potential increased. In Phase III, relatively narrow prediction interval widths were observed at both the low and high end of the observed range of irritation potential; wider intervals were observed in the middle of the observed range. In general, the selected endpoints in each phase had similar average prediction interval widths and thereby differed only slightly in their ability to predict MAS to a given level of precision; any differences between endpoints tended to occur at the low and/or high ends of the observed range of irritation potential. The primary contributor to total variability associated with prediction of MAS is the deviation between the Draize score as observed in the laboratory and what is predicted by the model for a given formulation. Consistently, this component is responsible for 70% to 95% of the total variability. The other components (i.e. variability among replicate MAS and in vitro scores) could be reduced simply by increasing the number of replicate tests performed on each test formulation. However, this would have relatively little impact on the overall precision of prediction.

A comparison of low volume, Draize and in vitro eye irritation test data. III. Surfactant-based formulations.

The third phase in a series of investigations of the relationship between low volume eye test (LVET) data, Draize eye irritation test data, and comparable data from in vitro assay protocols is presented. These investigations utilize Draize eye test and in vitro endpoint data generated previously as part of the CTFA Evaluation of Alternatives Program. LVET data were generated de novo using the same 25 representative surfactant-based personal-care formulations. In general, these formulations were minimally to moderately irritating. The linear correlation between maximum average score as determined by the Draize test (MAS) and the LVET (LVET-MAS) was 0.87; LVET-MAS values were typically about 30% lower then corresponding MAS values. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and in vitro endpoint. Regression modelling was the primary means of enabling such a comparison, the objective being to predict LVET-MAS for a given test material (and to place upper and lower prediction bounds on the range in which the LVET-MAS is anticipated to fall with high probability) based on observation of an in vitro score for that material. The degree of 95% confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Twenty in vitro endpoints were shown to have the greatest agreement with the LVET (these endpoints included those with low discordance rates relative to the Draize test) and were therefore selected for regression modelling. Although prediction interval widths tended to be narrower when predicting LVET-MAS compared with predicting MAS, the confidence with which the selected in vitro endpoints predicted both LVET-MAS and MAS for surfactant-based formulations was greatest when values were close to the lower or upper limits of the observed irritation range (i.e. 95% prediction interval widths were most narrow in these areas). Overall precision of LVET-MAS prediction for surfactant-based formulations was similar to that previously reported for hydroalcoholic formulations and considerably better than was reported for oil/water emulsions.

A comparison of low volume, draize and in vitro eye irritation test data. II. Oil/water emulsions.

The second phase in a series of investigations of the relationship between low volume eye test (LVET) data, Draize eye irritation test data, and comparable data from in vitro eye irritation test protocols is presented. These investigations utilize Draize eye test and in vitro endpoint data generated previously as part of the CTFA Evaluation of Alternatives Program. LVET data were generated de novo using the same 18 representative oil/water based personal-care formulations. In general, these formulations were minimally to mildly irritating; only three were classified as moderate eye irritants. The linear correlation between maximum average score as determined by the Draize test (MAS) and the LVET (LVET-MAS) was 0.85; LVET-MAS values were typically about half the corresponding MAS values. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and each in vitro endpoint. Regression modelling was the primary means of enabling such a comparison, the objective being to predict LVET-MAS for a given test material (and to place upper and lower 95% prediction bounds on the range in which the LVET-MAS is anticipated to fall with high probability) based on observation of an in vitro score for that material. The degree of confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Sixteen endpoints were shown to have the greatest agreement with the LVET (all but two were selected for modelling when compared with the Draize procedure). While the lower maximum average scores values (compared with the Draize test) in the LVET led to lower variability in LVET-MAS compared to MAS, the upper and lower bounds on predicted LVET-MAS values conditional on observed in vitro scores were still wide. Because there was overlap in the range of scores determined by the prediction bounds for many formulations, each of the selected endpoints was frequently unable to distinguish between test formulations in terms of statistically different predicted LVET-MAS values. In summary, none of the in vitro endpoints evaluated were able to reliably predict values of LVET-MAS among the set of oil/water emulsions considered here.

Percutaneous penetration of N-nitroso-N-methyldodecylamine through human skin in vitro: application from cosmetic vehicles.

The human skin penetration of N-nitroso-N-methyldodecylamine (NDOMA) from isopropyl myristate (IPM) and two vehicles representative of cosmetic/personal care formulations was determined in vitro. When applied as an infinite dose in IPM (1 microgram/microliter) the average total absorption over 48 hr was 0.10 +/- 0.01% of the applied dose (all data are expressed as means +/- SE). When applied as a finite dose in a representative oil-in-water emulsion formulation the average total absorption over 48 hr was 4.66 +/- 0.76% of the applied dose. When applied as a finite dose in a representative shampoo formulation for 10 min, followed by rinsing (to represent in-use exposure conditions), the average total absorption over 48 hr was 0.75 +/- 0.17% of the applied dose. Approximately 72% of the NDOMA in the applied shampoo formulation was removed by rinsing. The overall data indicated that NDOMA could penetrate the skin but that penetration was low. The rate and extent of absorption, however, could be affected by differences in the vehicle of application, time of exposure and whether the formulation is (and the conditions are designed to mimic) a rinse-off or leave-on product.

Percutaneous penetration of octyl salicylate from representative sunscreen formulations through human skin in vitro.

The human skin penetration of [14C]octyl salicylate from two representative sunscreen vehicles was determined in vitro. 3H-sucrose was incorporated into all formulations and provided a marker for membrane integrity. When applied as a finite dose in an oil-in-water emulsion vehicle containing 5% (w/w) octyl salicylate, the average total absorption of 14C over 48 hr was 0.65+/-0.16% of the applied dose (representing a total amount permeated of 1.58+/-0.36 microg/cm2). When applied as an infinite dose in the oil-in-water emulsion vehicle the average total absorption of 14C over 48 hr was 0.47+/-0.22% of the applied dose (representing a total amount permeated of 27.54+/-13.91 microg/cm2). When applied as a finite dose in a representative hydroalcoholic formulation containing 5% (w/w) octyl salicylate, the average total absorption of 14C over 48 hr was 0.59+/-0.09% of the applied dose (representing a total amount permeated of 1.58+/-0.25 microg/cm2). When applied as an infinite dose in the hydroalcoholic formulation the average total absorption of 14C over 48 hr was 0.23+/-0.05% of the applied dose (representing a total amount permeated of 11.28+/-2.55 microg/cm2). The penetration of [14C]salicylic acid [applied at a concentration of 2.7% (w/w), in the oil-in-water emulsion] was also determined. When applied as a finite dose the average total absorption of 14C over 48 hr was 1.14+/-0.23% of the applied dose (representing a total amount permeated of 1.65+/-0.39 microg/cm2). These results suggest that the in vitro human skin permeation of octyl salicylate is relatively low. The amounts of octyl salicylate and salicylic acid permeated when applied in similar vehicles were remarkably similar over 48 hr (1.58 microg/cm2 and 1.65 microg/cm2, respectively). This suggests the possibility that the 14C label appearing in the receptor fluid may, in both cases, represent salicylic acid. If this is the case, then it is possible that the amount of octyl salicylate permeating through the skin is much less than that suggested by the data obtained here. This supposition is, however, entirely speculative and has yet to be confirmed experimentally.

Comparison of low-volume, Draize and in vitro eye irritation test data. I. Hydroalcoholic formulations.

The first phase in a series of investigations of the relationship between low-volume eye test (LVET) data, Draize eye irritation test data, and comparable data from 25 in vitro assay protocols is presented. These investigations utilize Draize eye test and in vitro assay data generated previously as part of the Cosmetic, Toiletry and Fragrance Association (CTFA) Evaluation of Alternatives Program. LVET data were generated de novo using the same 10 representative hydroalcoholic personal-care formulations. The linear correlation between maximum average score (MAS) as determined by the Draize test and the LVET (LVET-MAS) was 0.93. Comparison of in vitro assay performance with that of the LVET was determined by statistical analysis of the relationship between LVET-MAS and in vitro endpoint. As in the CTFA program, regression modelling is the primary means of enabling such a comparison. The objective is to predict LVET-MAS for a given test material (and to place upper and lower prediction interval bounds in the range in which the LVET-MAS is anticipated to fall with high probability) conditional on observing an in vitro assay score for that material. The degree of confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves. Four assays [EYTEX MPA (membrane partition assay), HET-CAM (hen's egg test-chorioallantoic membrane HET-CAM) I, neutral red release and HET-CAM II] were shown to have the greatest agreement with the LVET. These assays were also among those with low discordance rates relative to the Draize test. Prediction of LVET-MAS values from experimentally determined in vitro scores was more accurate for hydroalcoholic formulations with lower rather than higher irritancy potential.

A toxicological review of topical exposure to white mineral oils.

White mineral oils have a long history of safe use by humans in orally ingested and topically applied products. A re-evaluation of the use of certain mineral hydrocarbons in the preparation of food items by regulators in the UK, however, has prompted additional safety studies and a critical assessment of the toxicological effects of white mineral oils. As white mineral oils are present in many topically applied drug and non-drug products, it is of interest to review the toxicological effects of mineral oil produced by this route of exposure. Specifically, the concern regarding the safety of white mineral oils has arisen, in part, from results of subchronic (e.g 90 day) feeding studies that reported the presence of granulomas in liver and histiocytosis in mesenteric lymph nodes of Fischer 344 rats after oral ingestion of select white mineral oils. In contrast to these subchronic oral studies, repeated topical exposure to white mineral oils has not been found to produce liver granulomas, histiocytosis in the mesenteric or other lymph nodes, or any local or systemic toxicity including tumour formation in Fischer 344 rates, C3H mice, New Zealand White rabbits or beagle dogs at similar or higher exposures (mg/kg/day). On the basis of these findings and reports on negligible epidermal penetration of topically applied white mineral oils, there is no evidence of any hazard identified for topical exposure to white mineral oils at any dose in multiple species. This conclusion is supported by the long and uneventful human use of white mineral oils in drug and non-drug topically applied products.

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase III) surfactant-based formulations.

The CTFA Evaluation of Alternatives Program is an evaluation of the relationship between data from the Draize primary eye irritation test and comparable data from a selection of promising in vitro eye irritation tests. In Phase III, data from the Draize test and 41 in vitro endpoints on 25 representative surfactant-based personal care formulations were compared. As in Phase I and Phase II, regression modelling of the relationship between maximum average Draize score (MAS) and in vitro endpoint was the primary approach adopted for evaluating in vitro assay performance. The degree of confidence in prediction of MAS for a given in vitro endpoint is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curve. Prediction intervals reflect not only the error attributed to the model but also the material-specific components of variation in both the Draize and the in vitro assays. Among the in vitro assays selected for regression modeling in Phase III, the relationship between MAS and in vitro score was relatively well defined. The prediction bounds on MAS were most narrow for materials at the lower or upper end of the effective irritation range (MAS = 0-45), where variability in MAS was smallest. This, the confidence with which the MAS of surfactant-based formulations is predicted is greatest when MAS approaches zero or when MAS approaches 45 (no comment is made on prediction of MAS > 45 since extrapolation beyond the range of observed data is not possible). No single in vitro endpoint was found to exhibit relative superiority with regard to prediction of MAS. Variability associated with Draize test outcome (e.g. in MAS values) must be considered in any future comparisons of in vivo and in vitro test results if the purpose is to predict in vivo response using in vitro data.

Percutaneous penetration of dimethylnitrosamine through human skin in vitro: application from cosmetic vehicles.

Human skin penetration of N-dimethylnitrosamine (DMN) from three vehicles has been determined in vitro. When applied as an infinite dose in isopropyl myristate (IPM, 1 microgram/microliter) the average total absorption over 48 hr was 2.6 +/- 1.2% of the applied dose (all data presented are expressed as means +/- standard errors). When applied as a finite dose in a representative oil-in-water emulsion vehicle the average total absorption over 48 hr was 4.0 +/- 0.3% of the applied dose. When applied as a finite dose in a representative shampoo vehicle for 10 min followed by rinsing (i.e. to represent in-use exposure conditions) the average total absorption over 48 hr was 1.1 +/- 0.1% of the applied dose. Approximately 72% of the DMN in the applied shampoo vehicle was removed by rinsing. There was considerable evaporative loss of DMN from the IPM and oil-in-water emulsion vehicles, such that absorption was complete within 3 hr of application. The overall data indicate that DMN can penetrate the skin rapidly but that in practice the amount actually available for penetration is significantly reduced by high permeant volatility. In contrast, application of N-nitrosodiethanolamine (NDELA) at a concentration of 1 microgram/microliter as an infinite dose generated an average total absorption over 48 hr of 23.6 +/- 6.4%, representing a total flux of 103.9 +/- 28.4 micrograms/cm2. In the case of NDELA, no evaporative loss was evident.

The CTFA Evaluation of Alternatives Program: an evaluation of in vitro alternatives to the Draize primary eye irritation test. (Phase II) oil/water emulsions.

The Cosmetic, Toiletry and Fragrance Association (CTFA) Evaluation of Alternatives Program is an evaluation of the relationship between Draize ocular safety test data and comparable data from a selection of in vitro tests. In Phase II, 18 representative oil/water-based personal-care formulations were subjected to the Draize primary eye safety test and 30 in vitro assay protocols (14 different types of in vitro endpoints were evaluated; the remainder were protocol variations). Correlation of in vitro with in vivo data was evaluated using analysis of sensitivity/specificity and statistical analysis of the relationship between maximum average Draize score (MAS) and in vitro endpoint. Regression modelling is the primary approach adopted in the CTFA Program for evaluating in vitro assay performance. The objective of regression analysis is to predict MAS for a given test material (and to place upper and lower prediction interval bounds on the range in which the MAS is anticipated to fall with high probability) conditional on observing an in vitro assay score for that material. The degree of confidence in prediction is quantified in terms of the relative widths of prediction intervals constructed about the fitted regression curves: the narrower the prediction interval, the more predictive of the Draize score is the in vitro test result. 16 assays were shown to have the greatest agreement with the Draize procedure and were therefore selected for regression analysis. Based on the magnitude of the 95% prediction bounds of each of the 16 selected assays over the range of test data, it may be inferred that prediction of MAS values from experimentally determined in vitro scores is more accurate for oil/water-based formulations with lower rather than higher irritancy potential. The assays selected for modelling in Phase II generally exhibited weaker relationships with MAS than those selected in Phase I (evaluated using hydroalcoholic formulations), even though several assays were common to both Phases.

Framework for validation and implementation of in vitro toxicity tests.

The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technologic developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial, and regulatory communities, is recommended. Test validation acceptance is contingent on broad buy-in by disparate groups in the scientific community--academics, industry, and government. This is best achieved by early and frequent communication among parties and agreement on common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction, and refinement alternatives in toxicity testing.

Percutaneous penetration of N-nitrosodiethanolamine through human skin (in vitro): comparison of finite and infinite dose applications from cosmetic vehicles.

N-Nitroso compounds (nitrosamines) have been detected at the parts per billion level in a wide variety of matrices including industrial chemicals, pharmaceuticals, and food. Although N-nitrosodiethanolamine (NDELA) may be detected as an impurity in some cosmetic products, studies on NDELA absorption through human skin have been limited. A study to determine the extent of NDELA absorption following topical application was therefore undertaken to assist in the proper assessment of risk following unintended exposure. NDELA absorption was measured in vitro through human cadaver skin using isopropyl myristate (IPM) and generic prototype personal-care formulations (sunscreen and shampoo) spiked with [14C]NDELA. When applied as a finite dose at a concentration of 0.06% or lower, NDELA absorption was found to be a linear function of concentration. Total absorption at 48 hr ranged from approximately 35 to 65% of the dose and was formulation dependent (IPM > shampoo > or = sunscreen). Absorption occurred relatively rapidly from all formulations and peak rates of absorption were seen within the first 5 hr from the IPM and shampoo formulations. When applied as an infinite dose, total NDELA absorption followed a different rank order (shampoo > or = IPM > sunscreen) and evidence of barrier damage was seen with the shampoo formulation.

Report of the Validation and Technology Transfer Committee of the Johns Hopkins Center for Alternatives to Animal Testing. Framework for validation and implementation of in vitro toxicity tests.

The development and application of in vitro alternatives designed to reduce or replace the use of animals, or to lessen the distress and discomfort of laboratory animals, is a rapidly developing trend in toxicology. However, at present there is no formal administrative process to organize, coordinate, or evaluate validation activities. A framework capable of fostering the validation of new methods is essential for the effective transfer of new technological developments from the research laboratory into practical use. This committee has identified four essential validation resources: chemical bank(s), cell and tissue banks, a data bank, and reference laboratories. The creation of a Scientific Advisory Board composed of experts in the various aspects and endpoints of toxicity testing, and representing the academic, industrial and regulatory communities, is recommended. Test validation acceptance is contingent upon broad buy-in by disparate groups in the scientific community-academics, industry and government. This is best achieved by early and frequent communication among parties and agreement upon common goals. It is hoped that the creation of a validation infrastructure composed of the elements described in this report will facilitate scientific acceptance and utilization of alternative methodologies and speed implementation of replacement, reduction and refinement alternatives in toxicity testing.

Assessment of the reproductive toxic potential of dermally applied 2-hydroxy-4-methoxybenzophenone to male B6C3F1 mice.

The potential of 2-hydroxy-4-methoxybenzophenone (HMB) to cause male reproductive toxicity was assessed in B6C3F1 mice. HMB was administered topically for 13 weeks (5 days/week) to groups of 10 mice each at dosages of 0, 10, 20, 100, or 400 mg/kg/day. Additional high dosage and control mice were also included and euthanized at interim time points to characterize the time course of any effects. After 91 days (or at interim periods) mice were euthanized and reproductive organ weights, cauda epididymal sperm concentration and proportion of motile and abnormal sperm, and testicular spermatid concentration were determined. Testicular histology was evaluated in fixed tissue. HMB treatment had no effect on body weight gain or any of the male reproductive parameters assessed at any time point. These results indicate that topically applied HMB has no reproductive toxic potential in male B6C3F1 mice at dosages as high as 400 mg/kg/day.

Evaluation of the cumulative (repeated application) eye irritation and corneal staining potential of FD&C yellow no. 5, FD&C blue no. 1 and FD&C blue no. 1 aluminium lake.

A protocol to evaluate the ocular irritation, staining, and embedding potential of FD&C colours (Yellow No. 5, Blue No. 1, Blue No. 1 Aluminium Lake) produced by repeated topical application to rabbit eyes is described. Test materials (3%, w/v in aqueous vehicle) were administered once daily, for a total of 21 days, to the conjunctival sac of the right eye of New Zealand White Rabbits (6 of each sex per group) at a dose volume of 30 microliters. Control animals (6 of each sex) received 30 microliters of the vehicle daily. All animals survived and were free of significant clinical signs of toxicity throughout the study. Ophthalmoscopic examinations revealed that all animals were free of abnormalities considered to be of clinical importance; all animals were free of significant signs of ocular irritation, staining and particle embedment. The results of this study support the safe use of these materials in consumer products intended for use in the eye area.

The biological interaction of cis- and trans-urocanic acid and DNA.

The potential for the cis and trans isomers of urocanic acid to produce DNA damage was measured by assays for DNA binding (32P-postlabeling assay), for induction of DNA repair (unscheduled DNA synthesis assay) and induction of mutations (Salmonella typhimurium and Escherichia coli plate-incorporation assays). These assays did not detect any evidence of a direct effect of either isomer of urocanic acid on DNA over a wide range of concentrations. These results suggest that neither isomer of urocanic acid alone, nor ultraviolet-irradiation of either cis or trans-urocanic acid produces significant DNA damage under conditions that permit cell survival.

Enhanced decomposition of oxyferrous cytochrome P450CIA1 (P450cam) by the chemopreventive agent 3-t-butyl-4-hydroxyanisole.

The efficacy of 2(3)-t-butyl-4-hydroxyanisole (BHA) and other chemicals as chemopreventive agents against chemically induced cancer or toxicity may involve direct modulation of cytochrome P450 activity. Direct interaction of BHA with cytochrome P450 was investigated using substrate-bound, oxyferrous cytochrome P450CIA1 either in a reconstituted system containing cytochrome P450CIA1, putidaredoxin, and putidaredoxin reductase with NADH as electron donor or in the absence of physiological electron donors. In the reconstituted system, BHA caused a concentration-dependent decrease in the production of 5-exo-hydroxycamphor and a substoichiometric increase in hydrogen peroxide production. However, BHA did not appreciably inhibit either NADH oxidation or oxygen utilization under conditions optimal for accumulation of oxyferrous cytochrome P450CIA1 during steady-state metabolism of camphor. In the absence of electron donor, BHA enhanced decomposition of the ternary oxyferrous substrate complex of cytochrome P450CIA1 without the formation of any apparent spectral intermediate(s). The rate of decomposition of the oxyferrous complex was pseudo-first order and was dependent upon the concentration of BHA present. Enhanced decomposition of the complex was not attributable to catalytic turnover of cytochrome P450CIA1 (i.e., acquisition of a second electron from an indeterminate source) since no appreciable metabolism of either camphor or BHA was observed. The enhanced decomposition was accompanied by a substoichiometric increase in hydrogen peroxide production, suggesting that BHA may facilitate four-electron reduction of molecular oxygen to water. These results indicate that BHA inhibits cytochrome P450 function, presumably by enhancing autoxidation of the substrate-bound oxyferrous complex.

Possible role of intracellular Ca2+ in the toxicity of phenformin.

Selective use of various mitochondrial Ca2+ transport inhibitors indicated that significant Ca2+ redistribution may occur during the isolation of mitochondria. Exposure of guinea-pig liver mitochondria to phenformin (beta-phenethylbiguanide) during the isolation procedure resulted in decreased mitochondrial Ca2+. Novel isolation conditions were developed to determine liver mitochondrial calcium content considered to reflect that in vivo. Administration of phenformin to rats and guinea-pigs resulted in decreased mitochondrial Ca2+. Decreased liver mitochondrial Ca2+ correlated inversely with raised blood lactate concentrations in the guinea-pig; 2-oxoglutarate, but not succinate oxidation, was inhibited in these mitochondrial preparations. A mechanism of action for phenformin-associated lactic-acidosis, attributable to impaired mitochondrial function arising from inactivation of Ca2+-sensitive, NAD+-dependent mitochondrial dehydrogenases (e.g. 2-oxoglutarate dehydrogenase) due to alteration in mitochondrial calcium content, is proposed.