Silica nanoparticles administered at the maximum tolerated dose induce genotoxic effects through an inflammatory reaction while gold nanoparticles do not.

While the collection of genotoxicity data and insights into potential mechanisms of action for nano-sized particulate materials (NPs) are steadily increasing, there is great uncertainty whether current standard assays are suitable to appropriately characterize potential risks. We investigated the effects of NPs in an in vivo Comet/micronucleus (MN) combination assay and in an in vitro MN assay performed with human blood. We also incorporated additional endpoints into the in vivo study in an effort to delineate primary from secondary mechanisms. Amorphous silica NPs (15 and 55 nm) were chosen for their known reactivity, while gold nano/microparticles (2, 20, and 200 nm) were selected for their wide size range and lower reactivity. DNA damage in liver, lung and blood cells and micronuclei in circulating reticulocytes were measured after 3 consecutive intravenous injections to male Wistar rats at 48, 24 and 4h before sacrifice. Gold nano/microparticles were negative for MN induction in vitro and in vivo, and for the induction of DNA damage in all tissues. Silica particles, however, caused a small but reproducible increase in DNA damage and micronucleated reticulocytes when tested at their maximum tolerated dose (MTD). No genotoxic effects were observed at lower doses, and the in vitro MN assay was also negative. We hypothesize that silica NPs initiate secondary genotoxic effects through release of inflammatory cell-derived oxidants, similar to that described for crystalline silica (quartz). Such a mechanism is supported by the occurrence of increased neutrophilic infiltration, necrosis, and apoptotic cells in the liver, and induction of inflammatory markers TNF-α and IL-6 in plasma at the MTDs. These results were fairly consistent between silica NPs and the quartz control, thereby strengthening the argument that silica NPs may act in a similar, thresholded manner. The observed profile is supportive of a secondary genotoxicity mechanism that is driven by inflammation.

Relevance of sensitization to occupational allergy and asthma in the detergent industry.

There exists considerable historic experience of the relationship between exposure and both the induction of sensitization and the elicitation of respiratory symptoms from industrial enzymes of bacterial and fungal origin used in a wide variety of detergent products. The detergent industry in particular has substantial experience of how the control of exposure leads to limitation of sensitization with low risk of symptoms. However, the experience also shows that there are substantial gaps in knowledge, even when the potential occupational allergy problem is firmly under control, and also that the relationship between exposure and sensitization can be hard to establish. The latter aspect includes a poor appreciation of how peak exposures and low levels of exposure over time contribute to sensitization. Furthermore, while a minority of workers develop specific IgE, essentially none appear to have symptoms, a situation which appears to contradict the allergy dogma that, once sensitized, an individual will react to much lower levels of exposure. For enzymes, the expression of symptoms occurs at similar or higher levels than those that cause induction. In spite of some knowledge gaps, medical surveillance programs and constant air monitoring provide the tools for successful management of enzymes in the occupational setting. Ultimately, the knowledge gained from the occupational setting facilitates the completion of safety assessments for consumer exposure to detergent enzymes. Such assessments have been proven to be correct by the decades of safe use both occupationally and in consumer products.

The toxicology and immunology of detergent enzymes.

Detergent enzymes have a very good safety profile, with almost no capacity to generate adverse acute or chronic responses in humans. The exceptions are the limited ability of some proteases to produce irritating effects at high concentrations, and the intrinsic potential of these bacterial and fungal proteins to act as respiratory sensitizers, demonstrated in humans during the early phase of the industrial use of enzymes during the 1960s and 1970s. How enzymes generate these responses are beginning to become a little clearer, with a developing appreciation of the cell surface mechanism(s) by which the enzymatic activity promotes the T-helper (T(H))-2 cell responses, leading to the generation of IgE. It is a reasonable assumption that the majority of enzyme proteins possess this intrinsic hazard. However, toxicological methods for characterizing further the respiratory sensitization hazard of individual enzymes remains a problematic area, with the consequence that the information feeding into risk assessment/management, although sufficient, is limited. Most of this information was in the past generated in animal models and in vitro immunoassays that assess immunological cross-reactivity. Ultimately, by understanding more fully the mechanisms which drive the IgE response to enzymes, it will be possible to develop better methods for hazard characterization and consequently for risk assessment and management.

The hair dyes PPD and PTD fail to induce a T(H)2 immune response following repeated topical application in BALB/c mice.

1,4-Phenylenediamine (PPD) and the structurally-related 1,4-toluenediamine (PTD) are frequently used oxidative hair dye precursors that can induce a delayed-type hypersensitivity reaction known as contact allergy. Very rare cases of Type 1 (IgE-mediated) allergic responses associated with PPD or PTD have been reported among hair dye users. As part of an effort to determine if repeated dermal exposure to the dyes could induce a T-helper-2 (T(H)2) response, we used a dermal exposure regimen in mice reported to identify a T(H)2 response. Ear swelling was evident at post-final exposure to PPD and PTD, indicating that an immune response was observed. However, cytokine mRNA after repeated topical exposure to these two chemicals showed no shift in the expression toward the typical T(H)2 cytokines interleukin (IL)-4 and IL-10 compared to the T(H)1 cytokine interferon (IFN)-γ. Consistent with these cytokine profiles, no concomitant increase in total serum IgE antibody titer or in B220+IgE+ lymphocytes in lymph nodes and skin application site skin was detected. In contrast, using an identical exposure regimen, animals topically exposed to the known respiratory (Type 1) allergen toluene 2,4-diisocyanate (TDI) showed significant expression of IL-4 and IL-10 mRNA compared to IFN? as well as an increase in total serum IgE and in B220+IgE+ cells in lymph nodes and skin application site. The data generated are consistent with the pattern of adverse reactions to hair dyes seen clinically, which overwhelmingly is of delayed rather than immediate-type hypersensitivity. Although current animal models have a limited ability to detect rare T(H)2 responses to contact allergens, the present study results support the view that exposure to hair dyes is not associated with relevant T(H)2 induction.

Chemical reactivity measurements: potential for characterization of respiratory chemical allergens.

Allergic diseases of the skin and respiratory tract resulting from exposure to low molecular weight chemicals remain important issues for consumer product development and occupational/environmental health. Widespread opportunities for exposure to chemical allergens require that there are available effective methods for hazard identification and risk assessment. In the search for new tools for hazard identification/characterization there has been interest in developing alternative methods that will reduce, refine or replace the need for animals. One approach that shows promise is based on the measurement of the peptide reactivity of chemicals; the potential to form stable associations with protein/peptide being a key requirement for the induction of sensitization. Recent investigations using these systems have focused primarily on skin sensitizing chemicals. However, there is interest in the possibility of exploiting these same experimental approaches to distinguish between different forms of chemical allergens - as individual materials are primarily associated with one or the other form of sensitization in humans. These investigations may also provide insight into why chemical sensitizers can differ in the form of allergic disease they will preferentially induce. These opportunities are surveyed here against a background of the immunobiology of allergic sensitization and current state-of-the-art approaches to measurement of peptide/protein reactivity.

Assessing the risk of type 1 allergy to enzymes present in laundry and cleaning products: evidence from the clinical data.

Microbial enzymes have been used in laundry detergent products for several decades. These enzymes have also long been known to have the potential to give rise to occupational type 1 allergic responses. A few cases of allergy among consumers using dusty enzyme detergents were reported in the early 1970s. Encapsulation of the enzymes along with other formula changes were made to ensure that consumer exposure levels were sufficiently low that the likelihood of either the induction of IgE antibody (sensitization) or the elicitation of clinical symptoms be highly improbable. Understanding the consumer exposure to enzymes which are used in laundry and cleaning products is a key step to the risk management process. Validation of the risk assessment conclusions and the risk management process only comes with practical experience and evidence from the marketplace. In the present work, clinical data from a range of sources collected over the past 40 years have been analysed. These include data from peer reviewed literature and enzyme specific IgE antibody test results in detergent manufacturers' employees and from clinical study subjects. In total, enzyme specific IgE antibody data were available on 15,765 individuals. There were 37 individuals with IgE antibody. The majority of these cases were from the 1970s where 23 of 4687 subjects (0.49%) were IgE positive and 15 of the 23 were reported to have symptoms of allergy. The remaining 14 cases were identified post-1977 for a prevalence of 0.126% (14/11,078). No symptoms were reported and no relationship to exposure to laundry and cleaning products was found. There was a significant difference between the pre- and post-1977 cohorts in that the higher rates of sensitization with symptoms were associated with higher exposure to enzyme. The clinical testing revealed that the prevalence of enzyme specific IgE in the population is very rare (0.126% since 1977). This demonstrates that exposure to these strong respiratory allergens via use of laundry and cleaning products does not lead to the development of sensitization and disease. These data confirm that the risk to consumers has been properly assessed and managed and support the concept that thresholds of exposure exist for respiratory allergy. Expansion of enzyme use into new consumer product categories should follow completion of robust risk assessments in order to continue ensuring the safe use of enzymes among consumers.

Tissue distribution of 20 nm, 100 nm and 1000 nm fluorescent polystyrene latex nanospheres following acute systemic or acute and repeat airway exposure in the rat.

Understanding tissue distribution and clearance of nanomaterials following different routes of exposure is needed for risk assessment. F344 female rats received single or multiple exposures to 20 nm, 100 nm or 1000 nm latex fluorospheres by intravenous (i.v.) injection or oral pharyngeal aspiration into the airways. The presence of fluorospheres in tissues was assessed up to 90-120 days after the final dose. Blood, perfusion fluid, bone marrow, brain, eyes, feces, gut, heart, kidney, liver, lung, muscle, skin, spleen, thymus, tongue, urine and uterus plus ovaries were collected for analysis. Liver, spleen and lung were the greatest tissue depots for all particles following i.v. injection. The proportion of 100 nm and 1000 nm but not 20 nm spheres significantly increased in the spleen over time. Lung was the greatest tissue depot for all particles following single or repeat airway exposure. Greater than 95% of 1000 nm spheres that were recovered were in the lung in contrast to 70-80% of 20 nm spheres or 89-95% of 100 nm spheres. All 3 sizes were found in gut or gut+feces 1-7 days after lung exposure. The thymus was the largest extra-pulmonary depot for the particles; up to 25% of recovered 20 nm particles were in the thymus up to 4 months after exposure compared to 6% of 100 nm particles and 1-3% of 1000 nm particles. A small proportion of 20 nm particles were detected in kidney following both acute and repeat airway exposure. Low numbers of particles were found in the circulation (blood, perfusion), bone marrow, brain, heart, liver and spleen but not in eye, muscle, skin, tongue, ovaries, uterus or urine. These data show that the tissue targets of nano- and micron-sized spheres are very similar whether exposure occurs systemically or via the airways while the proportion of particles in some tissues and tissue clearance varies based on particle size.

Chemical respiratory allergy and occupational asthma: what are the key areas of uncertainty?

There is increasing concern about the association of respiratory disease with indoor air quality and environmental atmospheric pollution. Associated with this is the fact that in many countries there has been a significant increase in the prevalence of asthma. Against this background there is a need to address the toxicological, occupational and public health problems associated with the ability of some chemicals to cause allergic sensitization of the respiratory tract and occupational asthma. By definition allergic sensitization of the respiratory tract to chemicals is dependent upon the stimulation of an adaptive immune response that leads to development of respiratory allergy and/or asthma. Although IgE antibody is associated typically with respiratory sensitization to protein allergens, there is less certainty about the role played by antibodies of this type in chemical respiratory allergy and occupational asthma. There are currently no validated or widely accepted methods/models for the identification and characterization of chemicals that have the potential to induce allergic sensitization of the respiratory tract. These and other areas of uncertainty were debated during the course of and following a two day Workshop. The primary purpose of the Workshop was to consider the important clinical and toxicological issues associated with chemical respiratory allergy, and to identify key questions that need to be answered if real progress is to be made.

Lack of type 1 sensitization to laundry detergent enzymes among consumers in the Philippines: results of a 2-year study in atopic subjects.

BACKGROUND: Enzymes have been safely used in laundry products for many years. The risk of developing adverse responses to enzymes in laundry detergents among consumers in countries where hand laundry predominates is expected to be low. OBJECTIVES: To understand how consumers in hand laundry markets used detergent products; to show that use of enzyme-containing detergents did not lead to sensitization in an atopic population with compromised skin; and to show that enzyme detergents did not have an adverse effect on skin condition. METHODS: Women in the rural Philippines were chosen since they do hand laundry for several hours a day, every day. The skin prick test (SPT) tested for the presence of IgE antibody to common aeroallergens and to enzymes in detergent product. Atopic women used enzyme-containing laundry bars for hand laundry and personal cleansing. They also used enzyme-containing laundry granules for hand laundry. All subjects were evaluated by SPT with enzymes over 2 years. Hand and body skin conditions were also evaluated. RESULTS: None of the 1,980 subjects screened for eligibility into the 2-year study were SPT positive to enzymes, including 655 women who used enzyme-containing detergent for up to 1 year. None of the subjects in the study developed IgE to the enzymes. Enzymes had no adverse effect on skin condition or on the development of erosions on the hands. CONCLUSIONS: The 2-year study confirms that enzymes are safe for use in laundry products at or below levels tested in the study even when used by atopic consumers under extremely harsh conditions.

Development of allergic antibody to an enzyme in a body lotion: results of an 18-month clinical study.

Use of enzymes in cosmetic products is novel and the safety of these products is not well understood. The safety of a prototype enzyme-containing body moisturizer lotion was tested via measures of skin compatibility and potential to induce protease-specific IgE antibody in a clinical study. Female, atopic subjects (n = 1,100) used body lotion containing 100 ppm protease (Y217L BPN') for 5 consecutive days per month, for 18 months. Regular lotion was used the remaining days of each month. Skin evaluation and skin prick tests (SPT) were conducted every 3 months. Measures of skin hydration were made in a subset of subjects at 3-month intervals: skin biopsies occurred at baseline and at the first 3-month timepoint. Serum from SPT positive subjects was tested for specific IgE in an immunoCAP assay. Clinical evaluation and histopathology showed no skin irritation and increased hydration of the skin over time. Three of 864 subjects completing the study developed IgE antibody to the enzyme: 1 subject after 6 months product use and 2 subjects after 15 months product use. A fourth subject was found with IgE antibody 3 months after study termination. None had allergic symptoms associated with product use. Intermittent exposure to a low level of protease enzyme in a body lotion led to the development of specific IgE antibody in 0.46% of subjects. While this study showed favorable skin compatibility of the protease containing lotion, the occurrence of allergic antibody to the enzyme was unacceptable for product commercialization.

Control of occupational asthma and allergy in the detergent industry.

OBJECTIVE: To provide an overview of how a comprehensive preclinical, clinical, and industrial hygiene program has been successfully used to control allergy and asthma to enzymes used in the detergent industry. DATA SOURCES: The author performed a PubMed and ToxLine search of English-language articles with the keywords enzymes, occupational allergy, occupational asthma, detergent, and detergent industry from January 1, 1995, to January 1, 2002. Scientific meeting abstracts, books, and industry association papers on allergy and asthma in the detergent industry were also reviewed. In addition, the practical experience of one major detergent company was included in the review. STUDY SELECTION: All published work on this topic was reviewed, and the work that discussed the key highlights of control of occupational allergy and asthma to enzymes used in the detergent industry was selected for this review. RESULTS: The detergent industry has developed guidelines for the safety assessment of enzymes, control of exposure to enzymes, and medical surveillance of enzyme-exposed workers. Because of these guidelines, occupational allergy and asthma to enzymes used in the detergent industry have become uncommon events. Cases of disease have been documented in some manufacturing sites that have had poor adherence to the guidelines. Those manufacturing sites that have adhered to the guidelines have had few cases of allergy and asthma to enzymes among exposed workers. A review of medical data from these sites has shown that workers who have developed IgE antibody to enzymes can continue to work with enzymes and remain symptom free. CONCLUSIONS: Occupational allergy and asthma to enzymes used in the detergent industry have been successfully controlled via the use of preclinical, clinical, and industrial hygiene safety programs designed to minimize sensitization to enzymes and development of disease. The basic principles of these programs can be applied to other industries where occupational allergy and asthma to proteins are common.

Occupational asthma and allergy in the detergent industry: new developments.

This review highlights the latest developments in the control of enzyme-induced occupational asthma and allergy (rhinitis and conjunctivitis) in the detergent industry. The industry has developed guidelines for the safe handling of enzymes in order to reduce the risk of occupational allergy and asthma. Those manufacturing facilities that follow all of the guidelines enjoy very low or no cases of asthma and allergy among workers exposed to enzymes. The key to the success of the management of enzyme-induced allergy and asthma is prospective surveillance for the development of enzyme-specific IgE antibody before the onset of allergic symptoms. This allows for continuing interventions to reduce exposures, so as to minimize or eliminate those associated with symptoms. Workers with IgE to enzymes can still continue to work in the industry symptom-free for their entire career. This indicates that exposures needed to induce sensitization are different and probably lower than exposures needed to elicit enzyme allergic symptoms. The experience of the detergent enzyme industry in controlling occupational allergens can be applied to other industries. The detergent enzyme story can be viewed as a model for the control of type 1 protein allergens in the workplace.