Safety evaluation of two α-amylase enzyme preparations derived from Bacillus licheniformis expressing an α-amylase gene from Cytophaga species.

A safety assessment was conducted for a symthetic variant Cytophaga sp. α-amylase enzyme expressed in Bacillus licheniformis and formulated into two distinct product formats: whole broth (a preparation in which the production organism is completely inactivated, but containing residual cell debris) and clarified preparation (from which the production organism is completely removed). The enzyme was improved via modern biotechnology techniques for use in the endohydrolysis of starch, glycogen, related polysaccharides and oligosaccharides. Applications range from carbohydrate processing, including the manufacture of sweeteners, fermentation to produce organic acids, amino acids and their salts, and potable or fuel alcohol, with resulting co-products (distillers' grains and corn gluten feed/meal) destined for use in animal feed. The toxicological studies summarized in this article (90-day rodent oral gavage and in vitro genotoxicity studies) noted no test article-related adverse effects and thus substantiate the safety of the α-amylase in not only the clarified form but also as a whole-broth preparation. Consistent with the decision tree analysis for enzymes produced with modern biotechnology techniques, this paper provides supporting information that this variant amylase with homology to an amylase from a potentially pathogenic organism (Cytophaga sp.) can be safely produced in an expression host that belongs to a Safe Strain Lineage, for safe use as processing aid to manufacture human and animal food.

Complete artificial saliva alters expression of proinflammatory cytokines in human dermal fibroblasts.

Complete artificial saliva (CAS) is a saliva substitute often used as a vehicle for test articles, including smokeless tobacco products. In the course of a study employing normal adult human dermal fibroblasts (HDFa) as a model in vitro, we discovered that CAS as a vehicle introduced a significant change in the expression of proinflammatory cytokines. To determine the effects of CAS on gene expression, real-time quantitative reverse-transcriptase PCR gene array analysis was used. Results indicate that robust changes in the expression of the proinflammatory cytokine interleukin 8 (IL8) and the vascular cell adhesion molecule 1 (VCAM1) occur within 5h of exposure to CAS. To determine whether CAS also alters cytokine release into the culture media, cytometric bead array assays for human inflammatory cytokines were performed. Analysis shows that CAS induced the release of IL8 and IL6. This study focused on determining which components in CAS were responsible for the proinflammatory response in HDFa. The following components were investigated: α-amylase, lysozyme, acid phosphatase, and urea. Results demonstrated that enzymatically active α-amylase induced gene expression for proinflammatory cytokines IL8, IL6, tumor necrosis factor-α, and IL1α and for VCAM1. Therefore, it is important to carefully evaluate the "vehicle effects" of CAS and its components in in vitro toxicology research.

What is the best strategy to reduce the burden of occupational asthma and allergy in bakers?

RATIONALE: Insight into the effectiveness of intervention strategies will help realise a decrease in the occupational disease burden from (allergic) respiratory diseases in the bakery population. OBJECTIVES: To use a simulation model to assess the impact of different intervention strategies on the disease burden of the bakery population over time. METHODS: A recently developed dynamic population based model was used to prospectively evaluate the impact on disease burden resulting from different intervention strategies. We distinguished interventions based on exposure reductions for flour dust and fungal α-amylase, health surveillance combined with reduction in exposure, and pre-employment screening. MAIN RESULTS: The impact of most interventions on disease burden was limited, generally less than 50% for lower respiratory symptoms and disabling occupational asthma. Only the rigorous health surveillance strategy, identifying workers who are sensitised or report upper respiratory symptoms and decreasing their individual exposures by 90% shortly after diagnosis, resulted in a decrease of almost 60% in disease burden after 20 years. CONCLUSIONS: This study demonstrates that different intervention strategies have substantially different impacts on the burden of disease. The time window during which changes occur differs considerably between strategies. This information can assist policy makers in their choice of intervention and gives guidance for achievable reductions in disease burden.

A dynamic population-based model for the development of work-related respiratory health effects among bakery workers.

OBJECTIVES: This paper presents a dynamic population-based model for the development of sensitisation and respiratory symptoms in bakery workers. The model simulates a population of individual workers longitudinally and tracks the development of work-related sensitisation and respiratory symptoms in each worker. METHODS: The model has three components: a multi-stage disease model describing the development of sensitisation and respiratory symptoms in each worker over time; an exposure model describing occupational exposure to flour dust and allergens; and a basic population model describing the length of a worker's career in the bakery sector and the influx of new workers. Each worker's disease state is modelled independently using a discrete time Markov Chain, updated yearly using each individual's simulated exposure. A Bayesian analysis of data from a recent epidemiological study provided estimates of the yearly transition probabilities between disease states. RESULTS: For non-atopic/non-sensitised workers the estimated probabilities of developing moderate (upper respiratory) symptoms and progression to severe (lower respiratory) symptoms are 0.4% (95% CI 0.3 to 0.5%) and 1.1% (95% CI 0.6 to 1.9%) per mg/m(3)/year of flour dust, respectively, and approximately twice these for atopic workers. The model predicts that 36% (95% CI 26 to 46%) of workers with severe symptoms are sensitised to wheat and 22% (95% CI 12 to 37%) to alpha-amylase. The predicted mean latency period for respiratory symptoms was 10.3 years (95% CI 8.3 to 12.3). CONCLUSIONS: While the model provides a valuable population-level representation of the mechanisms contributing to respiratory diseases in bakers, it was primarily developed for use in quantitative health impact assessment. Future research will use the model to evaluate a range of workplace interventions, including achievable reductions in exposure and health surveillance. The general methodology is applicable to other diseases such as chronic obstructive pulmonary disease, silicosis and musculoskeletal disorders and could be particularly valuable for forecasting changes in long latency diseases.

Modelling exposure in flour processing sectors in the Netherlands: a baseline measurement in the context of an intervention program.

INTRODUCTION: Recent studies have shown that even low exposure levels to flour dust and related allergens can cause severe respiratory symptoms. In The Netherlands the Dutch government and responsible branch organizations [from bakeries (traditional & industrial), flour mills and bakery ingredient producers] signed a covenant to reduce exposure to flour dust and decrease the prevalence of work-related occupational airway disease. This paper describes a sector wide survey to measure exposure to flour dust, wheat allergens and fungal alpha-amylase. The results are being used to underpin various elements of the covenant. METHODS: A dataset containing 910 personal measurements was compiled from four field studies containing information on exposure and potential determinants. The dataset represents a baseline estimate of exposure for four major flour processing sectors in The Netherlands. Exposure models for all sectors and agents were generated, based on job, tasks and company size, taking into account worker and company as random effect components. Use of control measures and, where possible, their effect were evaluated. RESULTS: Flour dust and enzyme exposures vary strongly between sectors. The job performed and specific tasks were identified as important determinants of exposure. The number of identified control measures during walk-through surveys, and their effectiveness in reduction of dust exposure was generally limited. The exposure models explained significant exposure variability between companies and workers but performed poorly in explaining day to day differences in exposure. DISCUSSION: The dataset serves as a baseline estimate and will be compared with a post intervention survey in the near future. The information obtained on control measures can be used to optimize the intervention scenarios that will be implemented in the different sectors by external occupational hygienists. The predictive exposure models will provide a relevant measure of average personal exposure that will be used in the sector wide health surveillance system.

Exposure-response relations for work related respiratory symptoms and sensitisation in a cohort exposed to alpha-amylase.

AIMS: To explore relations between exposure to fungal alpha-amylase and the risk of new work related respiratory symptoms or sensitisation. METHODS: A prospective cohort study among 300 bakers and millers was followed up for a maximum of seven years. Exposure to alpha-amylase was estimated by air measurements and questionnaires and classified into three categories. Symptoms were recorded with a self-administered questionnaire and skin sensitisation assessed using skin prick test (SPT). RESULTS: There were 36 new cases of chest symptoms, 86 of eyes/nose symptoms, and 24 of a positive SPT to alpha-amylase. There were exposure-response relations for chest and eyes/nose symptoms and for sensitisation, and a significantly increased prevalence ratio for chest symptoms in the highest exposure category. CONCLUSION: A reduction in alpha-amylase exposure is likely to reduce the risk for respiratory morbidity in bakery workers.

Exposure to inhalable dust, wheat flour and alpha-amylase allergens in industrial and traditional bakeries.

This study was designed to characterize exposure to inhalable dust, wheat flour and alpha-amylase allergens in industrial and traditional bakeries. The study included 70 bakeries from the northern part of Belgium. Based on the degree of automation and a clear division of individual job tasks, four bakeries were identified as industrial and the remaining 66 were identified as traditional ones. Personal, as well as stationary, samples of inhalable dust were collected during full shift periods, usually 5-7 h. The portable pumps aspirated 2 l/min through Teflon personal dust samplers (Millipore, pore size 1.0 microm) mounted in PAS-6 sampling heads. In the collected samples the inhalable dust, wheat flour and alpha-amylase allergens were determined. Wheat flour allergens were measured using enzyme-linked immunosorbent assay inhibition and an antiwheat IgG4 serum pool. The alpha-amylase allergens were measured using a sandwich enzyme immunoassay with affinity-purified polyclonal rabbit IgG antibodies. In total, 440 samples (300 personal and 140 stationary) were processed. The highest inhalable dust exposure was observed in traditional bakeries among bread [geometric mean (GM) 2.10 mg/m3] and bread and pastry workers (GM 1.80 mg/m3). In industrial bakeries the highest dust exposure was measured in bread-producing workers (GM 1.06 mg/m3). Similar relations were observed for wheat flour and alpha-amylase allergens. Bread baking workers in traditional bakeries had the highest exposure to both allergens (wheat flour GM 22.33 microg/m(3), alpha-amylase GM 0.61 ng/m3). The exposure to wheat flour and alpha-amylase allergens in industrial bakeries was higher in bread baking workers (wheat flour GM 6.15 microg/m3, alpha-amylase GM 0.47 ng/m3) than in bread packing workers (wheat flour GM 2.79 microg/m3, alpha-amylase GM 0.15 ng/m3). The data presented suggest that, on average, exposure in the Belgium bakeries studied-industrial as well as traditional-is lower than or similar to bakeries in The Netherlands, Canada, Sweden, the United Kingdom and Finland. Furthermore, the exposure levels in traditional bakeries seem to be higher than in industrial bakeries.

Safety evaluation of an alpha-amylase enzyme preparation derived from the archaeal order Thermococcales as expressed in Pseudomonas fluorescens biovar I.

BD5088 alpha-amylase derived from archaeal sources has characteristics of pH and temperature tolerance that are well suited to hydrolysis of starch in food processing applications. The production microorganism recipient strain, Pseudomonas fluorescens biovar I, strain MB101, was avirulent after oral administration to mice and does not represent an infectious threat to humans. Repeated dose gavage studies with BD5088 enzyme preparation, up to 13 weeks in duration, showed no systemic toxicity due to the oral route with an NOAEL of 890 mg/kg/day as Total Organic Solids. Some irritation occurred in the respiratory tract, which was considered to be a consequence of reflux and aspiration of test material that contained lipopolysaccharide from the Pseudomonas production strain. A 2-week dietary study (0 and 310 mg/kg/day) confirmed that there were no respiratory tract effects related to oral ingestion. There was no genotoxic activity based on Ames, mouse lymphoma, mouse micronucleus, and rat lymphocyte chromosomal aberration tests. There was no evidence of allergenic potential based on a comparison of the primary sequence of BD5088 with sequences in an allergen database. The enzyme was labile to pepsin digestion. Based on these data, BD5088 alpha-amylase preparation may be considered safe for use in food production such as corn wet milling.

Subchronic toxicity studies in dogs and in utero rats fed diets containing Bacillus stearothermophilus alpha-amylase from a natural or recombinant DNA host.

Beagle dogs and Fischer 344 rats were fed diets containing 0, 36 or 72 units Bacillus stearothermophilus alpha-amylase (Bsa)/g food or of Bacillus subtilis alpha-amylase (cBsa)/g food. The dogs (four/sex/group) received treated diets for 13 wk. For the rat studies, the parental (F0) generation (12 males and 24 females/group for the Bsa study, and 26 rats/sex/group for the cBsa study) received treated diets for 13 or 4 wk, respectively, before breeding and through weaning of the F1 pups; 20 F1 rats/sex/group received treated diets for at least 13 wk (from weaning until necropsy). There were no treatment-related antemortem observations, reproductive effects or ophthalmic, haematological, macroscopic or microscopic findings in treated dogs or rats, and no differences were noted in body weights for dogs or parental rats. Mean body weights of F1 pups from F0 rats exposed to 72 units cBsa/g were significantly lower than those of the control animals on lactation day 28. This effect may have been related to the slight reduction in body weights and significant reduction in food consumption (gestation days 14-20) of the F0 dams. However, this did not continue into the growth phase for the F1 generation. In the Bsa studies, there were no treatment-related effects in clinical pathology values, and organ-weight data did not correlate with macroscopic or microscopic findings. Male dogs given cBsa had significantly lower albumin (36 units/g), calcium (36 and 72 units/g) and inorganic phosphorus (72 units/g) values compared with those of the control males; there were no treatment-related changes in blood chemistry values in rats. Based on the results of these studies, the no-observable-effect level for alpha-amylase fed to dogs or rats is 36 units/g food.