Article

Evaluation of the genotoxicity of the food additive, gum ghatti

Authors:
  • Integrated Laboratory Systems, an Inotiv Company
  • Maronpot Consulting LLC, Raleigh, North Carolina
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Abstract

Gum ghatti is a food additive in some parts of the world, serving as an emulsifier, a stabilizer, and a thickening agent. To evaluate its genotoxic potential, we conducted Good Laboratory Practice compliant in vitro and in vivo studies in accordance with the Organisation for Economic Co-operation and Development (OECD) guidelines. No evidence of toxicity or mutagenicity was detected in a bacterial reverse mutation assay using five tester strains evaluating gum ghatti at up to 6 mg/plate, with or without metabolic activation. Gum ghatti also did not induce chromosome structural damage in a chromosome aberration assay using Chinese hamster ovary cells. To assess the ability to induce DNA damage in rodents, a combined micronucleus/Comet assay was conducted in male B6C3F1 mice. Gum ghatti was administered at 1000, 1500, and 2000 mg/kg/day by gavage once daily for 4 days and samples were collected 4h after the final dosing. No effect of gum ghatti was measured on micronucleated reticulocyte (MN-RET) frequency in peripheral blood, or DNA damage in blood leukocytes or liver as assessed by the Comet assay. Our results show no evidence of genotoxic potential of gum ghatti administered up to the maximum concentrations recommended by OECD guidelines.

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... Such sugar substitutes generally contain very few or no calories and therefore induce little or no glycemic response even though they taste like sugar. Sugar substitutes are now freely used in the production of processed foods like puddings, jellies, baked goods, jams, carbonated beverages, candy and powder drink mixes (Hobbs et al. 2012). ...
... Sorbitol (also called glucitol) is a sugar alcohol which undergoes a slow metabolism in human system. Diet foods, cough syrups, toothpaste, mints, sugar-free chewing gums and mouthwash are all sweetened with sorbitol (Hobbs et al. 2012). Although, sorbitol has a minimal influence on the glycemic level and few calories when compared with sugar, yet, consumption of large quantities of sorbitol can cause diarrhea (Shafrir 1991). ...
... Sorbitol has also been found to induce DNA fragmentation in Chinese Hamster ovary cells (Johannes et al. 1992). Regular consumption of additives in food induces cytotoxic, genotoxic and carcinogenicity (Zengin et al. 2011;Hobbs et al. 2012;Saad et al. 2014). Damage to the DNA by food additives is dependent on several factors among which are their transportation across the nuclear and/or nuclear membranes, deactivation and activation of processes involving intracellular enzymes, the level of scavenging radicals and the mechanism(s) of repair possessed by the exposed cells (Howard and Wylie-Rosete 2002). ...
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... FDA 2019) because of its emulsifying, stabilizing, and thickening properties. Gum ghatti is commercially used as a food additive and as a pharmaceutical excipient [5,6]. ...
... The oral toxicity potential of pure and modified gum was examined using a variety of biochemical and clinical test parameters, as well as histopathological analysis. No evidence of genotoxicity and mutagenicity was reported in earlier studies [6]. Maroupt et al. performed oral toxicity studies for 90 days, using gum ghatti in rat models [14]. ...
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Various drug delivery systems were developed using a modified form of gum ghatti. Modifying gum ghatti using thioglycolic acid improves its mucoadhesive property, and hence, it is a suitable approach for the fabrication and development of controlled drug delivery systems. In accordance with regulatory guidelines, namely, the Organization for Economic Co-operation and Development’s (OECD) 423 guidelines, an acute oral dose toxicity study was performed to examine the toxicological effects of gum ghattiin an animal (Wistar rat) after a single oral dose administration of pure gum ghatti and thiolated gum ghatti. Orally administered pure and thiolated gum ghatti do not reveal any considerable change in the behavioral pattern, food intake, body weight, hematology, or clinical symptoms of treated animals. Furthermore, histopathological studies showed no pathological mutations in the vital organs of Wistar rats after the oral administration of single doses of both types of gumghatti (i.e., 300 mg/kg and 2000 mg/kg body weight). Whole blood clotting studies showed the low absorbance value of the modified gum (thiolated gum ghatti) in contrast to the pure gum and control, hence demonstrating its excellent clotting capability. The aforementioned toxicological study suggested that the oral administration of a single dose of pure and thiolated gum ghatti did not produce any toxicological effects in Wistar rats. Consequently, it could be a suitable and safe candidate for formulating various drug delivery systems.
... These results were considered by the study authors to be incidental findings and not an indication of a true positive response. The results did not demonstrate any decrease in viable cell counts or mitotic index, or any increase in chromosome aberrations (as measured by the percentage of damaged cells in each culture) for any of the treatment cultures compared to vehicle controls (Swarts, 2010b; also reported in Hobbs et al., 2012). stated) on gestation days 6-15. ...
... The Committee considered this increase in caecal weight at 5% dietary gum ghatti to be an adaptive, rather than adverse, response. Based on the results of the new 90-day studies (Davis & Lea, 2011;Davis, 2012), the Committee identified a NOAEL of 3044 mg/kg bw per day (equal to 2590 mg/kg bw per day, corrected for purity), the highest dose tested. ...
... These results were considered by the study authors to be incidental findings and not an indication of a true positive response. The results did not demonstrate any decrease in viable cell counts or mitotic index, or any increase in chromosome aberrations (as measured by the percentage of damaged cells in each culture) for any of the treatment cultures compared to vehicle controls (Swarts, 2010b; also reported in Hobbs et al., 2012). stated) on gestation days 6-15. ...
... The Committee considered this increase in caecal weight at 5% dietary gum ghatti to be an adaptive, rather than adverse, response. Based on the results of the new 90-day studies (Davis & Lea, 2011;Davis, 2012), the Committee identified a NOAEL of 3044 mg/kg bw per day (equal to 2590 mg/kg bw per day, corrected for purity), the highest dose tested. ...
... It changes the behavior of water in food products and act as an emulsifier, stabilizer, and thickening agent. 81 Various properties of gum ghatti such as acid resistance, salt resistance, and ability to bind with oil are better than those of other gums. As compare to gum arabic, gum ghatti has a high potential against acid resistance, salt resistance, and the ability to bind with oil at lower concentration. ...
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Microwave (MW)-based dry blanching can inactivate oxidative enzymes like peroxidase (POD) and polyphenol oxidase (PPO) rapidly and retain a higher amount of water-soluble nutrients, like ascorbic acid. This study compared the MW-based dry blanching of potato slices of various thicknesses (5, 8, and 10 mm) with conventional methods (water and steam blanching). The time required for water and steam blanching was longer than that required for MW blanching. Potato slices of 10 mm thickness required a longer blanching duration compared with slices of a lesser thickness (5 and 8 mm). The MW-blanched samples (77.37–83.5%) retained a higher content of ascorbic acid, followed by steam-blanched (69.15–74.92%) and water-blanched (67.18–71.54%) samples. The Page, modified Page, Midilli–Kucuk, and Hii, Law, and Cloke models predicted the thin layer drying of potato slices (5 mm thickness) better with a higher coefficient of determination values (0.9607–0.9976) compared to Fick’s and Exponential models (0.8942–0.9444).
... It changes the behavior of water in food products and act as an emulsifier, stabilizer, and thickening agent. 81 Various properties of gum ghatti such as acid resistance, salt resistance, and ability to bind with oil are better than those of other gums. As compare to gum arabic, gum ghatti has a high potential against acid resistance, salt resistance, and the ability to bind with oil at lower concentration. ...
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Gum ghatti, popularly known as Indian gum and obtained from Anogeissus latifolia, is a complex high-molecular-weight, water-soluble, and swellable nonstarch polysaccharide comprised of magnesium and calcium salts of ghattic acids and multiple monosugars. Unlike other nontimber forest produce, gums ghatti is a low-volume but high-value product. It has several applications and is widely used as food, in pharmaceuticals, and for wastewater treatment and hydrogel formation, and it has attracted a great deal of attention in the fields of energy, environmental science, and nanotechnology. Industrial applications of gum ghatti are primarily due to its excellent emulsification, stabilization, thickening, heat tolerance, pH stability, carrier, and biodegradable properties. However, utilization of gum ghatti is poorly explored and implemented due to a lack of knowledge of its production, processing, and properties. Nevertheless, there has been interest among investigators in recent times for exploring its production, processing, molecular skeleton, and functional properties. This present review focuses on production scenarios, processing aspects, structural and functional properties, and potential applications in the food, pharmaceuticals, nonfood, and other indigenous and industrial usages.
... Thus, it is imperative that the consumer prevents the food products highest in sugar, having had in these last decades its substitution for intense sweeteners of low caloric value (Neacsu and Madar, 2014). However, the continuous alimentary additive ingestion has been associated with toxic, genotoxic and neoplastic effects (Demir et al., 2010;Hoobs et al., 2012;Saad et al., 2014). So, food with synthetic sweeteners, such as saccharin, cyclamate, aspartame and acesulfame potassium must be prevented (Durán et al., 2013;Thiyagarajan and Venkatachalam, 2012) For these reasons, the ingestion of natural sweeteners always must be preferential, since that the same one is in balanced dosages. ...
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... Previous studies have performed the following safety tests on gum ghatti: bacterial reverse mutation test, chromosome aberration test, and in vivo combined micronucleus/comet assays, which all revealed no genotoxicity (Hobbs et al., 2012). Furthermore, there were no toxicological findings in male and female rats after the 90-day administration of gum ghatti mixed in with feed at a maximum of 5% gum ghatti, resulting in an estimated no observed adverse effect level (NOAEL) of 3000-4000 mg/kg/day (Maronpot et al., 2013). ...
Article
We investigated the potential toxicity of gum ghatti, which is added to food for emulsifying, thickening, and stabilizing, after 4 weeks of repeated oral administration at a dose of 8000 mg/kg/day to male and female SD rats. Although food consumption was significantly reduced in males in the gum ghatti group compared with those in the distilled water group from Day 18 onwards, the change was minor, there was no pathological evidence of digestive tract abnormalities, and there were no significant changes in body weight; therefore, the change in food consumption was judged to be of no toxicological significance. Hematology and blood biochemistry revealed statistically significant differences in some parameters between the gum ghatti group and the distilled water group. These changes were all within the normal range of physiological variation and therefore were not considered to represent the effects of gum ghatti. In addition, general signs, body weight, and pathology showed no changes in either sex attributable to gum ghatti. Thus, all changes observed were of no toxicological significance and within the normal range of physiological variation, suggesting gum ghatti has no toxic effects in rats.
... Thus, it is imperative that the consumer prevents the food products highest in sugar, having had in these last decades its substitution for intense sweeteners of low caloric value (Neacsu and Madar, 2014). However, the continuous alimentary additive ingestion has been associated with toxic, genotoxic and neoplastic effects (Demir et al., 2010;Hoobs et al., 2012;Saad et al., 2014). So, food with synthetic sweeteners, such as saccharin, cyclamate, aspartame and acesulfame potassium must be prevented (Durán et al., 2013;Thiyagarajan and Venkatachalam, 2012) For these reasons, the ingestion of natural sweeteners always must be preferential, since that the same one is in balanced dosages. ...
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Stevia rebaudiana leaf extracts are calorie-free sweeteners of natural origin, derived from the Stevia rebaudiana plant known as a natural sweetener, which contains steviol glycosides and others bioactive compounds recognized by their biological properties. The present study was designed to evaluate the total phenolics (26.0 mg gallic acid/g) and total flavonoids contents (9.7 mg catechin/g) of a hydroalcoholic extract of Stevia rebaudiana dried leaves. A similar hydroalcoholic extract of commercial powder steviol sweetener was also evaluated, showing lower contents of bioactive compounds (11.9 mg/g and 5.1 mg/g, for total phenolics and flavonoids, respectively). The hydroalcoholic extract of dried Stevia rebaudiana leaves also showed high in vitro antioxidant activity, besides a positive correlation between total phenolic compounds and the DPPH and FRAP assays. Moreover, Stevia rebaudiana leaves have sensory and functional properties superior to those of many other high-potency sweeteners and is likely to become a major source of natural sweetener for the growing food market. Thus, four different lemon cakes formulations were studied (a traditional cake control recipe with sugar, two cakes with incorporation of Stevia rebaudiana fresh leaf and a cake with commercial powder steviol), using a sensory analysis covering 100 untrained consumers. Centesimal composition analyses of the four lemon cakes showed significant differences in fat, ashes, proteins and carbohydrates contents (p<0.05). Also, the raised energy value observed for the cake control was superior to the cake with Stevia rebaudiana leaves incorporation (309.8 Kcal/100 g, 268.0 Kcal/100 g,
... Chromosomal aberration assays using Chinese hamster ovary CHO- WBL cells were conducted as described previously ( Hobbs et al., 2012) with slight modifications to meet current OECD test guideline 473 (OECD, 1997a(OECD, , 2014b recommendations. Genipin and gardenia blue were formulated in DMSO and water, respectively; formulations were administered at 1% (DMSO) or 5% (water) of the final culture volume. ...
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Gardenia blue is widely used in Eastern Asia as a natural food colorant. To evaluate the genotoxic potential of gardenia blue, as well as genipin, the natural starting material from which it is produced, a GLP-compliant test battery was conducted according to OECD guidelines. No evidence of mutagenicity of gardenia blue was detected in a 5-strain bacterial reverse mutation assay, with or without metabolic activation; an equivocal response for genipin occurred in S. typhimurium TA97a without metabolic activation. In in vitro micronucleus and chromosome aberration assays, genipin tested positive under some test conditions; however, gardenia blue tested negative in both assays. In combined micronucleus/comet assays conducted in male and female B6C3F1 mice, exposure to genipin at doses reaching maximal toxicity (74 and 222 mg/kg bw/day for males and females, respectively) or gardenia blue tested up to the limit dose (2000 mg/kg bw/day) did not induce micronuclei in peripheral blood or DNA damage in several examined tissues. Modified ("reverse") comet assays showed no evidence of DNA crosslinking potential of either genipin, known to form crosslinks with other macromolecules, or gardenia blue. Our results indicate that consumption of gardenia blue in food products does not pose a significant genotoxic concern for humans.
... Allergy may cause severe symptoms and even a lifethreatening reaction (Gultekin and Doguc 2012). Some food additives have been shown to have a genotoxic effect and DNA damage on fetus (Hobbs et al. 2012). Nitrite and nitrate, used in processed food such as sausage and salami are known to turn into nitrosamines and cause cancer (Hill et al. 1973). ...
... However, their safety for human health and the environment has been controversial (Schiffman 2012;Suez et al. 2014;Di Luccia et al. 2015). Synthetic sweeteners could induce toxic, genotoxic, and carcinogenic effects as demonstrated for many food additives (Hobbs et al. 2012). The US Food and Drug Administration (FDA) banned cyclamate in 1970 from all dietary foods due to its potential to induce carcinogenesis in experimental animals. ...
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Low-calorie sweeteners are widespread. They are routinely introduced into commonly consumed food such as diet sodas, cereals, and sugar-free desserts. Recent data revealed the presence in considerable quantities of some of these artificial sweeteners in water samples qualifying them as a class of potential new emerging contaminants. This study aimed at evaluating the ecotoxicity profile of MNEI and Y65R-MNEI, two engineered products derived from the natural protein monellin, employing representative test organism such as Daphnia magna, Ceriodaphnia dubia, and Raphidocelis subcapitata. Potential genotoxicity and mutagenicity effects on Salmonella typhimurium (strain TA97a, TA98, TA100, and TA1535) and Escherichia coli (strain WP2 pkM101) were evaluated. No genotoxicity effects were detected, whereas slight mutagenicity was highlighted by TA98 S. typhimurium. Ecotoxicity results evidenced effects approximately up to 14 and 20% with microalgae at 500 mg/L of MNEI and Y65R-MNEI, in that order. Macrophytes and crustaceans showed no significant effects. No median effective concentrations were determined. Overall, MNEI and Y65R-MNEI can be classified as not acutely toxic for the environment.
... Although the CA is extensively employed in the toxicological genetics studies (ANDERSON; YU; MCGREGOR, 1998;ARALDI et al., 2014b;BURLINSON et al., 2007;HOBBS et al., 2012;LENT et al., 2012;VALDIGLESIAS et al., 2011), ...
... Specifically, these flavonols were evaluated in a bacterial reverse mutation assay ( Ames et al., 1975;Gatehouse et al., 1994;Maron and Ames, 1983) using Salmonella and E. coli tester strains and an in vitro MN assay (Avlasevich et al., 2011;Bryce et al., 2008) using the human TP53 competent TK6 lymphoblastoid cell line. In addition, as a thorough approach to assessing in vivo genotoxicity ( Pfuhler et al., 2007;Rothfuss et al., 2011), a combined MN/Comet assay was conduct- ed in male and female B6C3F1 mice ( Hobbs et al., 2012;Recio et al., 2010). The MN/Comet assay protocol used in our studies minimizes the use of experimental animals and complies with OECD Test Guideline 474 for the MN assay, as well as recommendations for the conduct of the Comet assay ( Burlinson et al., 2007;Tice et al., 2000). ...
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... Consistent ingestion of food additives has been reported to induce toxic, genotoxic, and carcinogenic effects (Demir et al., 2010;Hobbs et al., 2012;Güngörmüş and Kılıç, 2012;Jeffrey and Williams, 2000;Kumar and Srivastava, 2011;Saad et al., 2014Zengin et al., 2011. The DNA damage induced by food additives depends on their transport across cellular/nuclear membranes, the activation and deactivation of intracellular enzymatic processes, the levels of radical scavengers, and the repair mechanisms in the target cell population. ...
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Chapter
Gum ghatti is a green nonstarch polysaccharide that has seen significant interest in biomedical and pharmaceutical research because of its high viscosity, low cost, biodegradability, nontoxicity, and biocompatibility. Recently, several researchers have fabricated gum ghatti-based hydrogel for the delivery of drug and regeneration of tissue. This chapter discusses the general properties of gum ghatti and preparation of gum ghatti-based hydrogel. Moreover, applications of gum ghatti based-hydrogel in the delivery of drug and regeneration of tissue have been discussed.
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Resumo-Os aditivos alimentares se tornaram virtualmente obrigatórios na alimentação moderna, sobretudo por sua capacidade de manter a qualidade e a validade dos alimentos vendidos em supermercados. Entretanto, há estudos que associam a utilização inadequada desses componentes a efeitos prejudiciais à saúde, como o aparecimento de câncer, alergias e outras enfermidades. O objetivo do trabalho foi abordar o tema sobre os aditivos alimentares, suas aplicações e potencial toxicológico. A metodologia empregada foi o estudo exploratório descritivo através de pesquisa bibliográfica e da utilização de dados secundários oriundos de publicações e resultados de pesquisas específicas sobre o assunto. Observa-se que, de um ponto de vista tecnológico, os aditivos alimentares são considerados importantes por desempenhar papéis relevantes no processamento dos alimentos. Entretanto, seu uso desperta preocupação e questionamentos por parte dos consumidores. Verifica-se que foi determinada a genotoxicidade de 39 substâncias químicas utilizadas atualmente como aditivos alimentares. Sendo que de todos os aditivos, os corantes como amaranto, vermelho allura, new coccine, tartrazina, eritrosina, floxina e rosa bengala são exemplos de corante que induziram danos ao DNA relacionados com a dose glandular no estômago, cólon e/ou bexiga. Conclui-se que dentre as substâncias químicas utilizadas atualmente como aditivos alimentares os corantes foram considerados os mais genotóxicos, induzindo danos ao DNA. Abstract-The addictive ones alimentary if they turned virtually obligatory in the modern feeding, above all for his capacity to maintain the quality and the validity of the foods sold in supermarkets. The objective of the work was to approach the theme on the addictive ones alimentary, their applications and toxicology potential. The methodology was the exploratory-descriptive study through bibliographical research and of the use of secondary data originating from of publications and results of specific researches on the subject. Several sources were used as books, scientific goods, master's degree dissertations and legislations. Observed a technological point of view, food additives are considered important by playing an important role in food processing. However, it's use arouses concern and questions from consumers. It's noted that the genotoxicity of 39 chemicals presently used as food additives was determined. Being that all the additives, dyes like amaranth, allura red, new coccine, tartrazine, erythrosine, phloxine and rose bengal dye that are examples of induced DNA damage related to glandular dose in the stomach, colon and / or bladder. Concluded among the chemical substances used now as addictive alimentary, the colors were considered the more genotoxic, inducing damages to DNA.
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Abstract In vitro cytotoxicity assays can be used to evaluate potential toxicological effects of tobacco products. Total particulate matter (TPM) from mainstream cigarette smoke trapped by a Cambridge filter is used widely for biological evaluation of smoke. This present study compared neutral red uptake (NRU) assay, lactate dehydrogenase (LDH) activity assay and WST-1 assay for assessing the cytotoxicity of TPM, and evaluated the sensitivity of Chinese hamster ovary (CHO) cells and human lung adenocarcinoma epithelial cell line (A549 cells) to TPM-induced cytotoxic effects. The results indicate that NRU assay and WST-1 assay are preferable to LDH activity assay for assessing the TPM-induced cytotoxicity, and NRU assay might be more sensitive than WST-1 assay. The cytotoxicity of 3R4F reference cigarettes and two commercial brands of cigarettes were tested by NRU assay in CHO cells and A549 cells. The results showed that EC(50) values in CHO cells treated with TPM were lower than EC(50) values in A549 cells, indicating CHO cells are more sensitive to TPM-induced cytotoxic effects than A549 cells.
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The in vivo micronucleus (MN) assay has proven to be an effective measure of genotoxicity potential. However, sampling a single tissue (bone marrow) for a single indicator of genetic damage using the MN assay provides a limited genotoxicity profile. The in vivo alkaline (pH >13) Comet assay, which detects a broad spectrum of DNA damage, can be applied to a variety of rodent tissues following administration of test agents. To determine if the Comet assay is a useful supplement to the in vivo MN assay, a combined test protocol (MN/Comet assay) was conducted in male B6C3F1 mice and F344/N rats using four model genotoxicants: ethyl methanesulfonate (EMS), acrylamide (ACM), cyclophosphamide (CP), and vincristine sulfate (VS). Test compounds were administered on 4 consecutive days at 24-hr intervals (VS was administered to rats for 3 days); animals were euthanized 4 hr after the last administration. All compounds induced significant increases in micronucleated reticulocytes (MN-RET) in the peripheral blood of mice, and all but ACM induced MN-RET in rats. EMS and ACM induced significant increases in DNA damage, measured by the Comet assay, in multiple tissues of mice and rats. CP-induced DNA damage was detected in leukocytes and duodenum cells. VS, a spindle fiber disrupting agent, was negative in the Comet assay. Based on these results, the MN/Comet assay holds promise for providing more comprehensive assessments of potential genotoxicants, and the National Toxicology Program (NTP) is presently using this combined protocol in its overall evaluation of the genotoxicity of substances of public health concern.
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Since the publication of the International Programme on Chemical Safety (IPCS) Harmonized Scheme for Mutagenicity Testing, there have been a number of publications addressing test strategies for mutagenicity. Safety assessments of substances with regard to genotoxicity are generally based on a combination of tests to assess effects on three major end points of genetic damage associated with human disease: gene mutation, clastogenicity and aneuploidy. It is now clear from the results of international collaborative studies and the large databases that are currently available for the assays evaluated that no single assay can detect all genotoxic substances. The World Health Organization therefore decided to update the IPCS Harmonized Scheme for Mutagenicity Testing as part of the IPCS project on the Harmonization of Approaches to the Assessment of Risk from Exposure to Chemicals. The approach presented in this paper focuses on the identification of mutagens and genotoxic carcinogens. Selection of appropriate in vitro and in vivo tests as well as a strategy for germ cell testing are described.
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49 substances permitted for use in food in the United States was tested for mutagenicity in the Ames Salmonella typhimurium assay and in Escherichia coli strain WP2. Four of these substances caused increases in revertant counts in S. typhimurium. Two of these four (papain and pepsin) were found to contain histidine, and therefore the results of the tests on these two substances could not be taken as demonstrating mutagenicity. The other two substances causing increases in revertant counts (hydrogen peroxide and potassium nitrite) were mutagenic. The results on one chemical, beta-carotene, were evaluated as inconclusive or questionable. The remaining 44 substances were nonmutagenic in the test systems used. It is concluded that, for those generally physiologically innocuous chemicals tested, there are very few 'false positives' in the bacterial test systems used.
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The single cell gel electrophoresis (SCGE) or comet assay, which measures DNA strand breaks in individual cells, was used to analyse DNA damage and repair induced by the SN1-type alkylating carcinogens N-ethyl-N'-nitro-N-nitrosoguanidine and N-ethyl-N-nitrosourea in CHO cells. The comet assay was comparable in sensitivity to the alkaline elution assay. The alkyl-adducts detected as DNA single-strand breaks (ssb) by this technique were completely repaired within 24 h after treatment. These data indicate that long-lived lesions, such as alkylphosphotriesters, are not converted into ssb under the standard SCGE alkaline conditions (pH 13.5). The lesions revealed by the comet assay are mainly apurinic/apyrimidinic (AP) sites and breaks formed as intermediates in the base excision repair process of N-alkylpurines. When SCGE was performed at pH 12.5 instead of pH 13.5 a lower level of ssb was detected and these breaks were completely resealed within 2 h after treatment. These data suggest that different subsets of lesions are detected under different pH conditions. The SCGE combined with inclusion within the cells of endonuclease III revealed that a high portion of AP sites induced by alkylation damage were not converted into ssb by alkali. The level of endonuclease III-sensitive sites decreased as a function of the repair time and by 24 h after treatment no sites were left on the DNA. The use of this modified SCGE assay allows the estimation of the total amount of unrepaired AP sites present on DNA. Alkylation-induced ssb as detected by the comet assay should be regarded as an indicator of repair rate and balance more than a measure of actual DNA damage.
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The in vivo alkaline single cell gel electrophoresis assay, hereafter the Comet assay, can be used to investigate the genotoxicity of industrial chemicals, biocides, agrochemicals and pharmaceuticals. The major advantages of this assay include the relative ease of application to any tissue of interest, the detection of multiple classes of DNA damage and the generation of data at the level of the single cell. These features give the Comet assay potential advantages over other in vivo test methods, which are limited largely to proliferating cells and/or a single tissue. The Comet assay has demonstrated its reliability in many testing circumstances and is, in general, considered to be acceptable for regulatory purposes. However, despite the considerable data published on the in vivo Comet assay and the general agreement within the international scientific community over many protocol-related issues, it was felt that a document giving detailed practical guidance on the protocol required for regulatory acceptance of the assay was required. In a recent meeting held in conjunction with the 4th International Comet Assay Workshop (Ulm, Germany, 22-25 July 2001) an expert panel reviewed existing data and recent developments of the Comet assay with a view to developing such a document. This paper is intended to act as an update to the more general guidelines which were published as a result of the International Workshop on Genotoxicity Test Procedures. The recommendations are also seen as a major step towards gaining more formal regulatory acceptance of the Comet assay.
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Accurate measurement of low levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in DNA is hampered by the ease with which guanine is oxidized during preparation of DNA for analysis. ESCODD, a consortium of mainly European laboratories, has attempted to minimize this artifact and to provide standard, reliable protocols for sample preparation and analysis. ESCODD has now analyzed 8-oxodGuo in the DNA of lymphocytes isolated from venous blood from healthy young male volunteers in several European countries. Two approaches were used. Analysis of 8-oxodGuo by HPLC with electrochemical detection was performed on lymphocytes from 10 groups of volunteers, in eight countries. The alternative enzymic approach was based on digestion of DNA with formamidopyrimidine DNA glycosylase (FPG) to convert 8-oxo-7,8-dihydroguanine (8-oxoGua) to apurinic sites, subsequently measured as DNA breaks using the comet assay (7 groups of volunteers, in six countries). The median concentration of 8-oxodGuo in lymphocyte DNA, calculated from the mean values of each group of subjects as determined by HPLC, was 4.24 per 10(6) guanines. The median concentration of FPG-sensitive sites, measured with the comet assay, was 0.34 per 10(6) guanines. Identical samples of HeLa cells were supplied to all participants as a reference standard. The median values for 8-oxodGuo in HeLa cells were 2.78 per 10(6) guanines (by HPLC) and 0.50 per 10(6) guanines (by enzymic methods). The discrepancy between chromatographic and FPG-based approaches may reflect overestimation by HPLC (if spurious oxidation is still not completely controlled) or underestimation by the enzymic method. Meanwhile, it is clear that the true background level of base oxidation in DNA is orders of magnitude lower than has often been claimed in the past.
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The in vivo comet assay (single cell gel electrophoresis assay) in its alkaline version (pH >13) is being increasingly used in genotoxicity testing of substances such as industrial chemicals, biocides, agrochemicals, food additives and pharmaceuticals. Recommendations for an appropriate performance of the test using OECD guidelines for other in vivo genotoxicity tests have been published. In this review, we critically discuss the biological significance of comet assay effects in general and the status of the test in current strategies for genotoxicity testing. Examples for practical applications of the in vivo comet assay and potential consequences of positive and negative test results are given. The significance of comet assay results for hazard identification and risk assessment is discussed. In accordance with international guidelines for genotoxicity testing the in vivo comet assay is recommended for follow-up testing of positive in vitro findings. It is particularly useful as a tool for the evaluation of local genotoxicity, especially for organs/cell types which cannot easily be evaluated with other standard tests. A positive result in an appropriately performed in vivo comet assay indicates genotoxicity of the test compound in the tissue tested and gains particular significance when a mutagenic potential of the test compound has already been demonstrated in vitro. Such findings will have practical consequences in the risk assessment processes and further development of substances.
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Our objective was to study whether products of oxidative stress, such as hydrogen peroxide (H2O2), trans-2-hexenal, and 4-hydroxy-2-nonenal (HNE), cause DNA damage in genes, relevant for human colon cancer. For this, total DNA damage was measured in primary human colon cells and colon adenoma cells (LT97) using the single-cell gel electrophoresis assay, known as “Comet Assay.” APC, KRAS, and TP53 were marked in the comet images using fluorescence in situ hybridization (Comet FISH). The migration of APC, KRAS, or TP53 signals into the comet tails was quantified and compared to total DNA damage. All three substances were clearly genotoxic for APC, KRAS, and TP53 genes and total DNA in both types of cells. In primary colon cells, TP53 gene was more sensitive toward H2O2, trans-2-hexenal, and HNE than total DNA was. In LT97 cells, the TP53 gene was more sensitive only toward trans-2-hexenal and HNE. APC and KRAS genes were more susceptible than total DNA to both lipid peroxidation products but only in primary colon cells. This suggests genotoxic effects of lipid peroxidation products in APC, KRAS, and TP53 genes. In LT97 cells, TP53 was more susceptible than APC and KRAS toward HNE. Based on the reported gatekeeper properties of TP53, which in colon adenoma is frequently altered to yield carcinoma, this implies that HNE is likely to contribute to cancer progression. This new experimental approach facilitates studies on effects of nutrition-related carcinogens in relevant target genes.
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The science and technology of hydrocolloids used in food and related systems has seen many new developments and advances over recent years. This book presents the latest research from leading experts in the field. Some of the topics covered within this book include biochemical characterisation, the use of antibodies, immunostaining and enzyme hydrolysis, chemical and physicochemical characterization, engineering food microstructure, the role of biopolymers in the formation of emulsions and foams, hydrocolloids and health aspects. This book will be a useful reference for researchers and other professionals in industry and academia, particularly those involved directly with food science.
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This chapter describes the natural tree exudate gum from India. The exudate gum has a glassy appearance and the colour can vary from dark red to translucent white depending on the shape which can be either as a nodule or spiro. The chapter then reviews the structure and botanical source of gum ghatti. Technical information relating to its solubility, sugar composition, protein content, molecular weight, viscosity and emulsification performance are directly compared with gum arabic. Gum ghatti's current regulatory status together with various food and other applications are also described.
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Gum ghatti contains a soluble gum and an insoluble gel which partially dissolves on boiling. Maceration of the gel in water gives a perfectly stable dispersion which for practical purposes behaves as a solution. The viscosity of the gel dispersion is ca 10–30 times that of the soluble gum. The proportion of gel in four commercial batches of gum ghatti varied from 8 to 23 %. The viscosity of the whole gum dispersion depends largely on the proportion of gel. The viscosity of gum ghatti can be closely controlled by blending to a fixed proportion of gel.
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An in vivo method for screening drugs, food additives, and other chemicals that might cause chromosomal aberrations has been tested. It is reliable, easy, and very much more rapid than the traditional method.
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The Ames Salmonella/microsome mutagenicity assay (Salmonella test; Ames test) is a short-term bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances that can produce genetic damage that leads to gene mutations. The test employs several histidine dependent Salmonella strains each carrying different mutations in various genes in the histidine operon. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. When the Salmonella tester strains are grown on a minimal media agar plate containing a trace of histidine, only those bacteria that revert to histidine independence (his+) are able to form colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner.The Ames test is used world-wide as an initial screen to determine the mutagenic potential of new chemicals and drugs. The test is also used for submission of data to regulatory agencies for registration or acceptance of many chemicals, including drugs and biocides. International guidelines have been developed for use by corporations and testing laboratories to ensure uniformity of testing procedures.This review provides historical aspects of how the Ames was developed and detailed procedures for performing the test, including the design and interpretation of results.
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The short-term in vitro mammalian cell chromosome aberration test is used to assess potential genotoxic hazard of test substances. Mammalian cells are cultured in vitro, exposed to a test substance, harvested, and the frequency of asymmetrical structural chromosome aberrations is measured. Human peripheral blood lymphocytes do not normally divide. The assessment of the effects of cyclophosphamide on lymphocytes, stimulated to divide in whole blood cultures in vitro, is described. Procedures that are important in generating accurate results are emphasised, to avoid false positive results. The study design for a regulatory assay, the use of established cell lines, alternative methods of measuring cytotoxicity and analysis of results are included.
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The micronucleus test (m.t.) in vivo is a method devised primarily for screening chemicals for chromosome-breaking effects. The test substances are normally applied sub-acutely to small mammals, and the effect is read in direct smears from bone marrow. This testing procedure, developed by SCHMID and coworkers1~3~5~6 has a number of important advantages over the analysis of bone-marrow metaphase chromosomes. In technique of preparation as well as the reading of the slides, it is simpler and faster than chromosome analysis in the same material, but not at the expense of accuracy.
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311 chemicals were tested under code, for mutagenicity, in Salmonella typhimurium; 35 of the chemicals were tested more than once in the same or different laboratories. The tests were conducted using a preincubation protocol in the absence of exogenous metabolic activation, and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters. Some of the volatile chemicals were also tested in desiccators. A total of 120 chemicals were mutagenic or weakly mutagenic, 3 were judged questionable, and 172 were non-mutagenic. The remaining 16 chemicals produced different responses in the two or three laboratories in which they were tested. The results and data from these tests are presented.
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Results from the testing of 108 coded chemicals in Chinese hamster ovary (CHO) cells for the induction of chromosome aberrations and sister chromatid exchanges (SCEs) are presented. All chemicals were tested with and without exogenous metabolic activation, using protocols designed to allow testing up to toxic doses. Cell harvest times could also be extended if chemical-induced cell cycle delay was seen. Chromosome aberrations were induced by 43 of the chemicals, and 66 induced SCEs; 37 of the chemicals were positive for both endpoints.
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The methods for detecting carcinogens and mutagens with the Salmonella mutagenicity test were described previously (Ames et al., 1975b). The present paper is a revision of the methods. Two new tester strains, a frameshift strain (TA97) and a strain carrying an ochre mutation on a multicopy plasmid (TA102), are added to the standard tester set. TA97 replaces TA1537. TA1535 and TA1538 are removed from the recommended set but can be retained at the option of the investigator. TA98 and TA100 are retained. We discuss other special purpose strains and present some minor changes in procedure, principally in the growth, storage, and preservation of the tester strains. Two substitutions are made in diagnostic mutagens to eliminate MNNG and 9-aminoacridine. Some test modifications are discussed.
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Micronuclei induced in bone marrow erythroblasts by clastogenic chemicals are easily detected in peripheral blood. In mice treated with nitrogen mustard, 7,12 dimethylbenz(a)anthracene, or cyclophosphamide, the peak incidence of micronucleated polychromatic erythrocytes in peripheral blood was at least as great as the maximum incidence in bone marrow. In each case the peak incidence in blood occurred on the day following the peak incidence observed in bone marrow. Thus, for general genetic screening purposes, monitoring micronuclei in peripheral blood rather than in bone marrow smears provides at least equal sensitivity, offers greater simplicity in sample preparation and scoring, permits multiple sampling of treated animals, and may also facilitate automated scoring and human cytogenetic monitoring.
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At the International Workshop on the Standardisation of Genotoxicity Test Procedures, in Melbourne (27-28 February 1993), the current international guidelines for the correct conduct of bacterial mutation assays were considered, and the major differences between them were examined. An attempt was made to construct a scientifically based, internationally harmonized protocol. The main points of agreement were as follows. The consensus opinion was that there are currently insufficient data to justify a preference for either the preincubation or plate-incorporation methodologies as the initial test. Whichever method is used there was consensus agreement that the bacterial test battery should consist of S. typhimurium TA1537, TA1535, TA98 and TA100. There was also consensus that the 3 strains TA97a, TA97 and TA1537 could be used interchangeably. Although it was not possible to achieve a consensus, the majority of the working group members agreed that strains for the detection of mutagens acting specifically on AT base pairs should be routinely included within the test battery. These strains may be S. typhimurium TA102 or E. coli WP2 strains (WP2 pKM101 and WP2 uvrA or WP2 uvrA pkM101). With regard to study design it was universally agreed that 5 doses of test compound should be used in each experiment, and a majority agreement was obtained for 3 plates per dose. The use of 2 plates per dose is acceptable ONLY if the experiment is repeated. It is recommended that the negative controls may consist of solvent control alone provided that historical data are available to demonstrate lack of effect of the solvent in question. Positive control compounds should be included in all experiments, although the nature of these control compounds need not be specified in the guidelines. There was consensus agreement that for non-toxic freely soluble test agents, an upper limit of 5 mg/plate should be tested (5 microliters per plate for liquids). For insoluble or toxic compounds, the recommendations were the same as those for other in vitro tests (see appropriate paper). A consensus agreement was reached on the need to carry out further tests if equivocal results are obtained in the initial test, although it was generally agreed that the design of the repeat study should be left flexible. As there are little or no data to support the use of an exact repeat assay, a majority of the group recommended that negative results in the first test should be further investigated by either conducting a modified repeat (e.g. S9 titration) or by conducting the alternative methodology.(ABSTRACT TRUNCATED AT 400 WORDS)
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Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.
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Methylation and 13C NMR analyses were carried out on the high-arabinose, acidic heteropolysaccharide of gum ghatti and the products obtained on three successive, controlled Smith degradations. The side chains contained mainly 2-O- and 3-O-substituted Araf units. Of these the second degradation eliminated remaining alpha-Araf units, and their beta anomers became evident. The proportion of Galp units gradually increased in the form of nonreducing end- and Galp units, although 3,6-di-O- and 3,4,6-tri-O-substituted Galp units diminished. After three degradations groups with consecutive 3-O-substituted beta-Galp units were formed. The proportion of periodate-resistant 3-O- and 2,3-di-O-substituted Manp units was maintained. As a guide to side-chain structures in the polysaccharide, seven of the 10 free reducing oligosaccharide fractions (PC) present in the gum were isolated and examined (NMR, ESIMS, and sometimes methylation analysis). Characterized are alpha-Araf-(1 --> 2)-Ara and three Ara-containing oligosaccharide fractions containing 2-O- and 3-O-substituted units. These gave respectively, ESIMS molecular ions arising from Ara(2), beta-Araf oligosaccharides with four units, beta-Araf oligosaccharides with seven units, and Hex(2)-Ara(4). Alpha-Rhap-(1 --> 4)-GlcA, alpha-Rhap-(1 --> 4)-beta-GlcpA-(1 --> 6)-Gal, and alpha-Rhap-(1 --> 4)-beta-GlcpA-(1 --> 6)-beta-Galp-(1 --> 6)-Gal represented other side chains.
Article
As part of the Fourth International Workshop on Genotoxicity Testing (IWGT), held 9-10 September 2005 in San Francisco, California, an expert working group on the Comet assay was convened to review and discuss some of the procedures and methods recommended in previous documents. Particular attention was directed at the in vivo rodent, alkaline (pH >13) version of the assay. The aim was to review those protocol areas which were unclear or which required more detail in order to produce a standardized protocol with maximum acceptability by international regulatory agencies. The areas covered were: number of dose levels required, cell isolation techniques, measures of cytotoxicity, scoring of comets (i.e., manually or by image analysis), and the need for historical negative/positive control data. It was decided that a single limit dose was not sufficient although the required number of dose levels was not stipulated. The method of isolating cells was thought not to have a qualitative effect on the assay but more data were needed before a conclusion could be drawn. Concurrent measures of cytotoxicity were required with histopathological examination of tissues for necrosis or apoptosis as the "Gold Standard". As for analysing the comets, the consensus was that image analysis was preferred but not required. Finally, the minimal number of studies required to generate a historical positive or negative control database was not defined; rather the emphasis was placed on demonstrating the stability of the negative/positive control data. It was also agreed that a minimum reporting standard would be developed which would be consistent with OECD in vivo genotoxicity test method guidelines.
Article
Based on new scientific developments and experience of the regulation of chemical compounds, a working group of the Gesellschaft fuer Umweltmutationsforschung (GUM), a German-speaking section of the European Environmental Mutagen Society, proposes a simple and straightforward approach to genotoxicity testing. This strategy is divided into basic testing (stage I) and follow-up testing (stage II). Stage I consists of a bacterial gene mutation test plus an in vitro micronucleus test, therewith covering all mutagenicity endpoints. Stage II testing is in general required only if relevant positive results occur in stage I testing and will usually be in vivo. However, an isolated positive bacterial gene mutation test in stage I can be followed up with a gene mutation assay in mammalian cells. If this assay turns out negative and there are no compound-specific reasons for concern, in vivo follow-up testing may not be required. In those cases where in vivo testing is indicated, a single study combining the analysis of micronuclei in bone marrow with the comet assay in appropriately selected tissues is suggested. Negative results for both end points in relevant tissues will generally provide sufficient evidence to conclude that the test compound is nongenotoxic in vivo. Compounds which were recognized as in vivo somatic cell mutagens/genotoxicants in this hazard identification step will need further testing. In the absence of additional data, such compounds will have to be assumed to be potential genotoxic carcinogens and potential germ cell mutagens.
Article
Until recently, the in vivo erythrocyte micronucleus assay has been scored using microscopy. Because the frequency of micronucleated cells is typically low, cell counts are subject to substantial binomial counting error. Counting error, along with inter-animal variability, limit the sensitivity of this assay. Recently, flow cytometric methods have been developed for scoring micronucleated erythrocytes and these methods enable many more cells to be evaluated than is possible with microscopic scoring. Using typical spontaneous micronucleus frequencies reported in mice, rats, and dogs we calculate the counting error associated with the frequency of micronucleated reticulocytes as a function of the number of reticulocytes scored. We compare this counting error with the inter-animal variability determined by flow cytometric scoring of sufficient numbers of cells to assure that the counting error is less than the inter-animal variability, and calculate the minimum increases in micronucleus frequency that can be detected as a function of the number of cells scored. The data show that current regulatory guidelines allow low power of the test when spontaneous frequencies are low (e.g., < or =0.1%). Tables and formulas are presented that provide the necessary numbers of cells that must be scored to meet the recommendation of the International Working Group on Genotoxicity Testing that sufficient cells be scored to reduce counting error to less than the inter-animal variability, thereby maintaining a more uniform power of detection of increased micronucleus frequencies across laboratories and species.
Article
The development of automated flow cytometric (FCM) methods for evaluating micronucleus (MN) frequencies in erythrocytes has great potential for improving the sensitivity, reproducibility, and throughput of the traditional in vivo rodent MN assay that uses microscopy-based methods for data collection. Although some validation studies of the FCM evaluation methods have been performed, a comprehensive comparison of these two data collection methods under routine testing conditions with a variety of compounds in multiple species has not been conducted. Therefore, to determine if FCM evaluation of MN frequencies in rodents was an acceptable alternative to traditional manual scoring methods in our laboratory, we conducted a comparative evaluation of MN-reticulocyte (MN-RET) frequencies determined by FCM- and microscopy-based scoring of peripheral blood and bone marrow samples from B6C3F1 mice and Fisher 344 rats. Four known inducers of MN (cyclophosphamide, ethyl methanesulfonate, vincristine sulfate, acrylamide) were assayed in bone marrow and peripheral blood of both mice and rats. In addition, MN-RET frequencies were measured in bone marrow (microscopy) and peripheral blood (FCM) of mice treated with five nongenotoxic chemicals (S-adenosylmethionine chloride, cefuroxime, diphenolic acid, 3-amino-6-methylphenol, pentabromodiphenyl oxide). No significant differences were observed between results obtained by the two methods in either species. These results support the use of FCM for determining MN-RET frequency in rodents after chemical exposure.
Article
The dye exclusion test is used to determine the number of viable cells present in a cell suspension. It is based on the principle that live cells possess intact cell membranes that exclude certain dyes, such as trypan blue, Eosin, or propidium, whereas dead cells do not. In this test, a cell suspension is simply mixed with dye and then visually examined to determine whether cells take up or exclude dye. In the protocol presented here, a viable cell will have a clear cytoplasm whereas a nonviable cell will have a blue cytoplasm.
International Validation of the in vivo Rodent Alkaline Comet Assay for the detection of genotoxic carcinogens testing protocol
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