Bacterial Mutation Assay Research Articles

Selection of solvent and positive control concentration for enhanced Ames test conditions for N-nitrosamine compounds

The Ames test is a widely used bacterial mutagenicity assay to evaluate the potential of chemical compounds to induce mutations. In recent years, there has been growing concern regarding the presence of N-nitrosamines in pharmaceuticals, food, and other consumer products. N-Nitrosamines are probable mutagens and carcinogens. To address the reduced sensitivity of the standard Ames test for N-nitrosamines, particularly N-nitrosodimethylamine, the European Medicines Agency (EMA) and United States Food and Drug Administration (FDA) have recently published recommendations for enhanced Ames test (EAT) conditions. However, there is a lack of clear guidance on the selection of N-nitrosamine positive control concentrations, particularly for 1-cyclopentyl-4-nitrosopiperazine, and the amount of solvent to be used in the EAT. This study aims to address the current gap in concentration and volume specifications by providing a comprehensive guide to set up enhanced Ames test conditions specifically for N-nitrosamine compounds using appropriate amounts of solvent, new solvents, and strain-specific positive control concentrations.

Leaching of unexpected cyazofamid degradation products into groundwater demonstrates gaps in current pesticide risk assessment

To investigate the formation and leaching potential of degradation products N,N-dimethylsulfamide (DMS) and dimethylsulfamic acid (DMSA) from cyazofamid under real-world agricultural conditions, the fungicide cyazofamid was applied in a potato crop as part of the Danish Pesticide Leaching Assessment Programme (PLAP). Leaching of DMS, DMSA, 4-chloro-5-(4-methylphenyl)-1H-imidazole-2-carbonitrile (CCIM), and 4-chloro-5-(4-methylphenyl)-1H-imidazole-2-carboxylicacid (CTCA) was monitored in water from the variably saturated zone (suction cups) and groundwater for more than two years following the applications. In total, 424 samples were analyzed for the content of the four degradation products. An additional laboratory study was executed in parallel with the field monitoring study. Here, cyazofamid was applied to soil columns and leaching of the four degradation products was studied under controlled conditions.In the EFSA conclusion on cyazofamid, CCIM and CTCA are mentioned as major relevant metabolites; DMS is not mentioned in the risk assessment and DMSA is only included in acute oral toxicity studies and an in vitro bacterial mutation assay. In contrast to the EFSA conclusion on cyazofamid, our studies showed no leaching of the two major metabolites, CTCA and CCIM, but instead, major leaching of DMS and DMSA in both the field and laboratory studies was observed. That is, both DMS and DMSA leached to the groundwater in concentrations >0.1 μg/L for more than half a year. Based on this, we suggest improvements to the current pesticide risk assessment.

Scientific opinion on flavouring group evaluation 415 (FGE.415): (E)-3-benzo[1,3]dioxol-5-yl-N,N-diphenyl-2-propenamide.

The EFSA Panel on Food Additives and Flavourings (FAF) was requested to evaluate the safety of the substance (E)‐3‐benzo[1,3]dioxol‐5‐yl‐N,N‐diphenyl‐2‐propenamide [FL‐no: 16.135] as a new flavouring substance, in accordance with Regulation (EC) No 1331/2008. The substance has not been reported to occur naturally and it is chemically synthesised. It is intended to be used as a flavouring substance in specific categories of food, but not intended to be used in beverages. The chronic dietary exposure to [FL‐no: 16.135] estimated using the added portions exposure technique (APET), is calculated to be 780 μg/person per day for a 60‐kg adult and 480 μg/person per day for a 15‐kg 3‐year‐old child. [FL‐no: 16.135] did not show genotoxic effects in bacterial mutagenicity and mammalian cell micronucleus assays in vitro. Developmental toxicity was not observed in a study in rats at the dose levels up to 1,000 mg/kg body weight (bw) per day. The Panel derived a BMDL of 101 mg/kg bw per day from a 90‐day toxicity study. Based on this BMDL, adequate margins of exposure of 7,800 and 3,200 could be calculated for adults and children, respectively. The Panel concluded that there is no safety concern for [FL‐no: 16.135], when used as a flavouring substance at the estimated level of dietary exposure calculated using the APET approach, based on the intended uses and use levels as specified in Appendix B. The Panel further concluded that the combined exposure to [FL‐no: 16.135] from its use as a food flavouring substance and from its presence in toothpaste is also not of safety concern.

The common indoor air pollutant α-pinene is metabolized to a genotoxic metabolite α-pinene oxide

1. α-Pinene caused a concentration-responsive increase in bladder hyperplasia and decrease in sperm counts in rodents following inhalation exposure. Additionally, it formed a prospective reactive metabolite, α-pinene oxide. 2. To provide human relevant context for data generated in animal models and explore potential mechanism, we undertook studies to investigate the metabolism of α-pinene to α-pinene oxide and mutagenicity of α-pinene and α-pinene oxide. 3. α-Pinene oxide was formed in rat and human microsomes and hepatocytes with some species differences. Based on area under the concentration versus time curves, the formation of α-pinene oxide was up to 4-fold higher in rats than in humans. 4. While rat microsomes cleared α-pinene oxide faster than human microsomes, the clearance of α-pinene oxide in hepatocytes was similar between species. 5. α-Pinene was not mutagenic with or without induced rat liver S9 in Salmonella typhimurium or Escherichia coli when tested up to 10,000 μg/plate while α-pinene oxide was mutagenic at ≥25 μg/plate. 6. α-Pinene was metabolized to α-pinene oxide under the conditions of the bacterial mutation assay although the concentration was approximately 3-fold lower than the lowest α-pinene oxide concentration that was positive in the assay, potentially explaining the lack of mutagenicity observed with α-pinene.

Safety Evaluation of Bifidobacterium lactis BL-99 and Lacticaseibacillus paracasei K56 and ET-22 in vitro and in vivo.

Probiotics have been reported to play a major role in maintaining the balance of microbiota in host. Consumption of food with probiotics has increased with consumer concerns regarding healthy diets and wellness. Correspondingly, safety evaluation of probiotics for human consumption has become increasingly important in food industry. Herein, we aimed to test the safety of Bifidobacterium lactis BL-99 and Lacticaseibacillus paracasei K56 and ET-22 strains in vitro and in vivo. In results, these strains were found to be negative for mucin degradation and platelet aggregation test. Additionally, the three strains were susceptible to eight antibiotics. In accordance with bacterial reversion mutation (Ames) assay, the tested strains had no genetic mutagenicity. Finally, it was confirmed that there were no dose-dependent mortality and toxicity throughout multidose oral toxicity tests in rats. Our findings demonstrated that B. lactis BL-99 and L. paracasei K56 and ET-22 can achieve the generally recognized as safe (GRAS) status as probiotics in the future.

Takashi Sugimura: In Memoriam (1926–2020)–A Personal Perspective

![][1] Takashi Sugimura was a pioneer in cancer research and a figure recognized internationally for his seminal contributions to the understanding of mutagenicity and chemical carcinogenesis. Rather than focusing on the many accolades, honors, and influential positions that he held,

Evaluation of 2-methoxy-4-nitroaniline (MNA) in hypersensitivity, 14-day subacute, reproductive, and genotoxicity studies

2-Methoxy-4-nitroaniline (MNA), an intermediate in the synthesis of azo dyes used in textiles and paints, is structurally similar to carcinogenic anilines. Human exposure occurs primarily in the occupational setting through handling of dye dust, and through use and disposal of MNA-containing products. MNA has been reported to induce contact hypersensitivity in a human, myocardial necrosis in rats, and bacterial mutagenicity. This study assessed the subacute toxicity, genotoxicity, contact hypersensitivity, and reproductive toxicity of MNA in rodents in an effort to more fully characterize its toxicological profile. B6C3F1/N mice were exposed to 0, 650, 1250, 2500, 5000, or 10,000 ppm MNA by dosed feed for 14-days to evaluate subacute toxicity and histopathological endpoints. In female mice, decreased body weight (13.5 %) and absolute kidney weight (14.8 %), compared to control, were observed at 10,000 ppm MNA; increased relative liver weight (10–12 %), compared to control, occurred at 5,000–10,000 ppm MNA. In male mice, absolute (15 %) and relative liver weights (9–13 %) were increased at 2,500−5,000 ppm and 1250–10,000 ppm MNA, compared to control, respectively. In both sexes of mice, minimal elevations of hemosiderin pigmentation (a breakdown product of erythrocytes), relative to control, were observed in the liver (10,000 ppm); minimal to moderate elevations of hemosiderin pigmentation (5,000–10,000 ppm) and minimal increases in hematopoietic cell proliferation occurred in the spleen (≥ 1250 ppm). In a reproductive toxicity study, timed-mated female Harlan Sprague Dawley rats were exposed to 0–10,000 ppm MNA by dosed feed from gestation day 6 through postnatal day (PND) 21. Decreases in mean litter weights were observed at 5000 ppm MNA, compared to control, beginning at PND1. To evaluate potential contact hypersensitivity, MNA (2.5–50 %, in dimethylformamide) was applied to the dorsa of both ears of female Balb/c mice once daily for three days. The increase observed in lymph node cell proliferation (10–50 % increase in thymidine uptake compared to control) did not reproducibly achieve the Sensitization Index (SI) 3 level, and there was no ear swelling evident following sensitization with 10–50 % MNA and challenge with 25 % MNA in the mouse ear swelling test. In bacterial mutagenicity assays, MNA (250−1000 μg/plate) induced significant increases, compared to control, in mutant colonies with and without metabolic activation enzymes in Salmonella typhimurium strains TA100 and TA98. These data indicate that MNA is genotoxic, and may induce erythrocyte damage and reactive phagocytosis by macrophages in the liver and spleen.

Preclinical safety assessment of pathogen reduced red blood cells treated with amustaline and glutathione.

ABSTRACTBACKGROUNDThe nucleic acid targeted pathogen reduction (PR) system utilizing amustaline (S‐303) and glutathione (GSH) is designed to inactivate blood‐borne pathogens and leukocytes in red blood cell concentrates (PR‐RBCC). Inactivation is attained after amustaline intercalates and forms covalent nucleic acid adducts preventing replication, transcription, and translation. After pathogen inactivation, amustaline spontaneously hydrolyzes to S‐300, the primary negatively charged reaction product; amustaline is below quantifiable levels in PR‐RBCC. GSH quenches free unreacted amustaline.STUDY DESIGN AND METHODSThe genotoxic and carcinogenic potential of PR‐RBCC, the reaction by‐products, and S‐300 were assessed in accordance with the International Conference on Harmonization (ICH) guidelines and performed in compliance with the Food and Drug Administration (FDA) good laboratory practice standards, 21 CFR Part 58. in vitro bacterial reverse mutagenicity and chromosomal aberration assays were performed with and without exogenous S9 metabolic activation, and in in vivo clastogenicity and carcinogenic assays using validated murine models.RESULTSPR‐RBCCs were not genotoxic in vitro and in vivo and were non‐carcinogenic in p53+/− transgenic mice transfused over 26 weeks. Estimated safety margins for human exposure ranged from >90 to >36 fold for 2 to 5 PR‐RBCCs per day, respectively. PR‐RBCCs and S‐300 did not induce chromosome aberration in the in vivo murine bone marrow micronucleus assay at systemically toxic doses.CONCLUSIONSPR‐RBCCs did not demonstrate genotoxicity in vitro or in vivo and were not carcinogenic in vivo. These studies support the safety of PR‐RBCCs and suggest that there is no measurable genotoxic hazard associated with transfusion of PR‐RBCCs.

Revisiting the bacterial mutagenicity assays: Report by a workgroup of the International Workshops on Genotoxicity Testing (IWGT)

The International Workshop on Genotoxicity Testing (IWGT) meets every four years to obtain consensus on unresolved issues associated with genotoxicity testing. At the 2017 IWGT meeting in Tokyo, four sub-groups addressed issues associated with the Organization for Economic Cooperation and Development (OECD) Test Guideline TG471, which describes the use of bacterial reverse-mutation tests. The strains sub-group analyzed test data from >10,000 chemicals, tested additional chemicals, and concluded that some strains listed in TG471 are unnecessary because they detected fewer mutagens than other strains that the guideline describes as equivalent. Thus, they concluded that a smaller panel of strains would suffice to detect most mutagens. The laboratory proficiency sub-group recommended (a) establishing strain cell banks, (b) developing bacterial growth protocols that optimize assay sensitivity, and (c) testing “proficiency compounds” to gain assay experience and establish historical positive and control databases. The sub-group on criteria for assay evaluation recommended that laboratories (a) track positive and negative control data; (b) develop acceptability criteria for positive and negative controls; (c) optimize dose-spacing and the number of analyzable doses when there is evidence of toxicity; (d) use a combination of three criteria to evaluate results: a dose-related increase in revertants, a clear increase in revertants in at least one dose relative to the concurrent negative control, and at least one dose that produced an increase in revertants above control limits established by the laboratory from historical negative controls; and (e) establish experimental designs to resolve unclear results. The in silico sub-group summarized in silico utility as a tool in genotoxicity assessment but made no specific recommendations for TG471. Thus, the workgroup identified issues that could be addressed if TG471 is revised. The companion papers (a) provide evidence-based approaches, (b) recommend priorities, and (c) give examples of clearly defined terms to support revision of TG471.

Inhibitory effect of purple rice husk extract on AFB1-induced micronucleus formation in rat liver through modulation of xenobiotic metabolizing enzymes

BackgroundRice husk, a waste material produced during milling, contains numerous phytochemicals that may be sources of cancer chemopreventive agents. Various biological activities of white and colored rice husk have been reported. However, there are few comparative studies of the cancer chemopreventive effects of white and colored rice husk.MethodsThis study investigated the cancer chemopreventive activities of two different colors of rice husk using in vitro and in vivo models. A bacterial mutation assay using Salmonella typhimurium strains TA98 and TA100 was performed; enzyme induction activity in murine hepatoma cells was measured, and a liver micronucleus test was performed in male Wistar rats.ResultsThe white rice husk (WRHE) and purple rice husk (PRHE) extracts were not mutagenic in Salmonella typhimurium TA98 or TA100 in the presence or absence of metabolic activation. However, the extracts exhibited antimutagenicity against aflatoxin B1 (AFB1) and 2-amino-3,4 dimethylimidazo[4,5-f]quinolone (MeIQ) in a Salmonella mutation assay. The extracts also induced anticarcinogenic enzyme activity in a murine Hepa1c1c7 hepatoma cell line. Interestingly, PRHE but not WRHE exhibited antigenotoxicity in the rat liver micronucleus test. PRHE significantly decreased the number of micronucleated hepatocytes in AFB1-initiated rats. PRHE contained higher amounts of phenolic compounds and vitamin E than WRHE in both tocopherols and tocotrienols as well as polyphenol such as cyanidin-3-glucoside, protocatechuic acid and vanillic acid. Furthermore, PRHE increased CYP1A1 and 1A2 activities while decreasing CYP3A2 activity in the livers of AFB1-treated rats. PRHE also enhanced various detoxifying enzyme activities, including glutathione S-transferase, NAD(P)H quinone oxidoreductase and heme oxygenase.ConclusionsPRHE showed potent cancer chemopreventive activity in a rat liver micronucleus assay through modulation of phase I and II xenobiotic metabolizing enzymes involved in AFB1 metabolism. Vitamin E and phenolic compounds may be candidate antimutagens in purple rice husk.

Demonstrating laboratory proficiency in bacterial mutagenicity assays for regulatory submission

The bacterial reverse mutation test is a mainstay for evaluation of mutagenicity predicting the carcinogenic potential of a test substance and is recommended by regulatory agencies across the globe. The popularity of the test is due, in part, to the relatively low cost, rapid results and small amount of test material required compared to most other toxicological tests as well as the near universal acceptance of the toxicological significance of a clear positive or negative result. Most laboratories follow the Organization for Economic Cooperation and Development Test Guideline 471 (TG471) or national guidelines based on TG471. Regulatory agencies in most countries are obligated to consider results from tests which meet the recommendations laid out in TG471. Nonetheless, laboratories unfamiliar with the test sometimes have trouble generating reliable, reproducible results. TG471 is a test guideline, not a detailed test protocol. A group of experts from regulatory agencies and laboratories which use the assay has assembled here a set of recommendations which if followed, will allow an inexperienced laboratory to acquire proficiency in assay conduct. These include recommendations for how to create a cell bank for the 5 Salmonella typhimurium/Escherichia coli strains and develop a laboratory protocol to reliably culture each strain to ensure each culture has the characteristics which allow adequate sensitivity for detection of mutagens using the test as described in TG471. By testing compounds on the provided lists of positive and negative test substances, the laboratory will have surmounted many of the problems commonly encountered during routine testing of unknown chemicals and will have gained the experience necessary to prepare the detailed protocol needed for performing the test under Good Laboratory Procedures and the laboratory will have generated the historical positive and negative control databases which are needed for test reports which adhere to TG471.

Assessment of Mutagenicity of Herbal Preparations from Al-Baha Region, Saudi Arabia

Background and Objective: Traditional use of herbal medicinal plants in Saudi Arabia is common and in particular in Al-Baha region either in rural or urban areas for treatment of several ailments and no reports have been found about the mutagenicity of Saudi medicinal plants. Here we investigated the mutagenic/genotoxic effects of four herbal plants methanolic extracts namely, Acalypha fruticosa, Aloe vera, Jatropha spp and Ricinus communis, using the Ames test Methodology: For assessment of the mutagenicity of these four plants, we used the bacterial reverse mutagenicity assay (Ames test) without any metabolic activation system. The plants methanolic extract concentrations used were 0.1, 1.0 and 5.0 mg/ml. Results: Low concentrations of the extracts (0.1 mg/ml) of the four plants were found to be non-mutagenic (p < 0.05), while only the extracts of Aloe vera and Jatropha were found to be mutagenic in the second concentration (1.0 mg/ml). Meanwhile, Acalypha fruticosa extract at 1.0 mg/ml was found to be non-mutagenic and Ricinu communis was found to be cytotoxic at this concentration. At the concentration of 5.0 mg/ml the three extracts (Acalypha fruticosa, Aloe vera, Jatropha ) were mutagenic. Conclusion: Only higher concentrations, 1.0 and 5.0 mg/ml extracts of the herbal plants Acalypha fruticosa, Aloe vera and Jatropha, were found to be mutagenic/genotoxic in the reverse bacterial mutagenicity Ames test.

Purification and interactions of the MucA\u2019 and MucB proteins constituting the DNA polymerase RI

BackgroundThe MucA’ and MucB proteins comprise the core of DNA polymerase RI which is a strong mutator utilized in mutagenicity assays such as the standard Ames test. A close relative DNA polymerase V, composed of the homologous UmuD’ and UmuC proteins, is considered to be an ortholog of the mammalian DNA polymerase η. The catalytic subunits of these polymerases belong to the Y-family which specializes in the translesion DNA synthesis across various DNA adducts to rescue stalled chromosomal replication at the expense of mutations. Based on genetic evidence, DNA polymerase RI possesses the greatest ability to induce various types of mutations among all so far characterized members of the Y-superfamily. The exceptionally high mutagenic potential of MucA’B has been taken advantage of in numerous bacterial mutagenicity assays incorporating the conjugative plasmid pKM101 carrying the mucAB operon such as the Ames Test.ResultsWe established new procedures for the purification of MucB protein as well as its accessory protein MucA’ using the refolding techniques. The purified MucA’ protein behaved as a molecular dimer which was fully stable in solution. The soluble monomeric form of MucB protein was obtained after refolding on a gel-filtration column and remained stable in a nondenaturing buffer containing protein aggregation inhibitors. Using the surface plasmon resonance technique, we demonstrated that the purified MucA’ and MucB proteins interacted and that MucB protein preferentially bound to single-stranded DNA. In addition, we revealed that MucB protein interacted with the β-subunit of DNA polymerase III holoenzyme of E. coli.ConclusionThe MucA’ and MucB proteins can be isolated from inclusion bodies and solubilized in vitro. The refolded MucB protein interacts with its MucA’ partner as well as with DNA what suggests it retains biological activity. The interaction of MucB with the processivity subunit of DNA polymerase III may imply the role of the subunit as an accessory protein to MucB during the translesion DNA synthesis.

Managing emerging mutagenicity risks: Late stage mutagenic impurity control within the atovaquone second generation synthesis

The mutagenic-impurity control strategy for a second generation manufacturing route to the non-mutagenic antipneumocystic agent atovaquone (2-((1R,4R)-4-(4-chlorophenyl)cyclohexyl)-3-hydroxynaphthalene-1,4-dione) 1 is described. Preliminary assessment highlighted multiple materials of concern which were largely discharged either through returning a negative bacterial mutagenicity assay or through confidence that the impurity would be purged during the downstream processing from when it was first introduced. Additional genotoxicity testing highlighted two materials of concern where initial assessment suggested that testing for these impurities at trace levels within the drug substance would be required. Following a thorough review of process purging detail, spiking and purging experimentation, and an understanding of the process parameters to which they were exposed an ICH M7 Option 4 approach could be justified for their control. The development of two 1H NMR spectroscopy methods for measurement of these impurities is also described as well as a proposed summary table for describing the underlying rationale for ICH M7 control rationales to regulators. This manuscript demonstrates that process purging of potential mutagenic impurities can be realised even when they are introduced in the later stages of a process and highlights the importance of scientific understanding rather than relying on a stage-counting approach.

ProTox-II: a webserver for the prediction of toxicity of chemicals.

Advancement in the field of computational research has made it possible for the in silico methods to offer significant benefits to both regulatory needs and requirements for risk assessments, and pharmaceutical industry to assess the safety profile of a chemical. Here, we present ProTox-II that incorporates molecular similarity, pharmacophores, fragment propensities and machine-learning models for the prediction of various toxicity endpoints; such as acute toxicity, hepatotoxicity, cytotoxicity, carcinogenicity, mutagenicity, immunotoxicity, adverse outcomes pathways (Tox21) and toxicity targets. The predictive models are built on data from both in vitro assays (e.g. Tox21 assays, Ames bacterial mutation assays, hepG2 cytotoxicity assays, Immunotoxicity assays) and in vivo cases (e.g. carcinogenicity, hepatotoxicity). The models have been validated on independent external sets and have shown strong performance. ProTox-II provides a freely available webserver for in silico toxicity prediction for toxicologists, regulatory agencies, computational and medicinal chemists, and all users without login at http://tox.charite.de/protox_II. The webserver takes a two-dimensional chemical structure as an input and reports the possible toxicity profile of the chemical for 33 models with confidence scores, and an overall toxicity radar chart along with three most similar compounds with known acute toxicity.

Black cohosh extracts and powders induce micronuclei, a biomarker of genetic damage, in human cells.

Black cohosh extract (BCE) is a widely used dietary supplement marketed to women to alleviate symptoms of gynecological ailments, yet its toxicity has not been well characterized. The National Toxicology Program (NTP) previously reported significant increases in micronucleated erythrocytes in peripheral blood of female Wistar Han rats and B6C3F1/N mice administered 15-1,000 mg BCE/kg/day by gavage for 90 days. These animals also developed a dose-dependent nonregenerative macrocytic anemia characterized by clinical changes consistent with megaloblastic anemia. Both micronuclei (MN) and megaloblastic anemia can arise from disruption of the folate metabolism pathway. The NTP used in vitro approaches to investigate whether the NTP's test lot of BCE, BCEs from various suppliers, and root powders from BC and other cohosh species, were genotoxic in general, and to gain insight into the mechanism of action of BCE genotoxicity. Samples were tested in human TK6 lymphoblastoid cells using the In Vitro MicroFlow® MN assay. The NTP BCE and a BC extract reference material (XRM) were tested in the MultiFlow® DNA Damage assay, which assesses biomarkers of DNA damage, cell division, and cytotoxicity. The NTP BCE and several additional BCEs were tested in bacterial mutagenicity assays. All samples induced MN when cells were grown in physiological levels of folic acid. The NTP BCE and BC XRM produced activity patterns consistent with an aneugenic mode of action. The NTP BCE and five additional BCEs were negative in bacterial mutagenicity tests. These findings show that black cohosh preparations induce chromosomal damage and may pose a safety concern. Environ. Mol. Mutagen. 59:416-426, 2018. © 2018 Published 2018. This article is a US Government work and is in the public domain in the USA.

Weight of contribution of in vitro chromosomal aberration assay for evaluation of pesticides: Experience of risk assessment at the Food Safety Commission of Japan

Due to the course of registration of pesticides in Japan, the Food Safety Commission (FSC) has the responsibility to make a risk assessment of residual pesticides and related chemicals through foods. Among the set of safety evaluations for pesticides, genotoxicity assay data are mandatory. The standard test battery for this evaluation consists of a bacterial gene mutation assay, in vitro mammalian chromosomal aberrations and/or other chromosome damage assay, and in vivo rodent micronucleus assay. These assay outcomes are used for mechanistic consideration of carcinogenicity, if any. As a rule, if a certain substance is carcinogenic and the mechanism of it includes genotoxicity, the FSC might decide it is not possible to establish the acceptable daily intake of that pesticide. Therefore, the information about genotoxicity is critical for potentially carcinogenic chemicals, whether the applied substance will be adopted and permitted for use or not as pesticides. It is important to assess fairly, carefully, and transparently, but feasible, rapid, and efficient assessment also should be taken into account. Therefore, needless to say, the assay(s) should have the sensitivity to detect potent mutagens. It is also important to be aware that the required data set should be consisted of reliable assays without certain assay(s) that give(s) false positive information or offer less of a contribution for the safety assessment.

Extreme testing of undiluted e-cigarette aerosol in vitro using an Ames air-agar-interface technique

There is a growing consensus that e-cigarettes hold the potential for reducing the harm associated with cigarette smoking. Recently published studies have reported in vitro testing of e-cigarettes, demonstrating reduced toxicological and biological effects. Few studies however have reported the use of e-cigarettes under extreme testing conditions. To assess the full mutagenic potential of a commercially available electronic-cigarette (Vype ePen), this study investigated the delivery of aerosol under extreme conditions, using a scaled-down 35 mm plate Ames bacterial reverse mutagenicity assay. S. typhimurium strains TA98, TA100, TA97, TA104 and E. coli WP2 uvrA pKM101 with or without metabolic activation (S9), were employed. Using a modified Vitrocell VC 10 exposure system 0, 180, 360, 540, 720 or 900 puffs of undiluted e-cigarette aerosol was generated and delivered to bacterial cultures aligned to reported human consumption data. The results demonstrate that no mutagenic activity was observed in any strain under any test condition even when exposed to 900 puffs of undiluted e-cigarette aerosols +/− S9. Positive control responses were observed in all strains +/− S9. Nicotine assessments demonstrated an increased and consistent aerosol delivery, with calculated maximum doses of ∼1 mg/mL delivery of nicotine. These data demonstrate the validity of this unique testing approach and adds further information to the growing weight of evidence that e-cigarettes offer substantially reduced exposure when compared to conventional cigarette smoke. For future in vitro assessments of next generation tobacco and nicotine products, the generation, delivery and testing of undiluted aerosols can now be considered.

Scientific Opinion of Flavouring Group Evaluation 410 (FGE.410): 4',5,7-trihydroxyflavanone from chemical group 25 (phenol derivatives containing ring-alkyl, ring-alkoxy, and side-chains with an oxygenated functional group).

The Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids (CEF) of EFSA was requested to deliver a scientific opinion on the implications for human health of the flavouring substance 4′,5,7‐trihydroxyflavanone or naringenin [FL‐no: 16.132], in the Flavouring Group Evaluation 410 (FGE.410), according to Regulation (EC) No 1331/2008 of the European Parliament and of the Council. The substance occurs naturally in grapefruits, oranges and tomatoes. It is intended to be used as a flavouring substance with flavour‐modifying properties in specific categories of food. Information on specifications and manufacturing of [FL‐no: 16.132] were considered adequate; however, data on stability in food are incomplete. The Panel noted that the available genotoxicity studies have significant shortcomings and are insufficient to conclude on the genotoxic potential of naringenin. Therefore, [FL‐no: 16.132] cannot be evaluated through the Procedure. Additionally, the Panel noted that inhibition of CYP 450 by [FL‐no: 16.132] has been clearly demonstrated in animal species in vivo which implies that the substance may interact with the metabolism and elimination of medicines and no convincing information is available that this does not pose a risk to humans at the estimated levels of exposure. To continue with the safety assessment of [FL‐no: 16.132], a bacterial gene mutation assay and an in vitro micronucleus assay (according to OECD guidelines 471, 487 and GLP) are required. Even if these studies do not indicate a genotoxic potential, additional toxicological data are needed to finalise the evaluation.

Genotoxicity and sub-chronic toxicity of MYOLUTION® (branched chain keto acids)

MYOLUTION®, which consists of a mixture of the branched chain keto acids, keto-leucine, keto-isoleucine and keto-valine, as their calcium salts, may potentially be used as a food ingredient based on the reported contributions of these compounds to muscle health and exercise performance. Tests on genotoxicity and sub-chronic toxicity were performed to evaluate the safety of branched chain keto acids. No genotoxic effects were observed in the bacterial mutation assay or the in vitro micronucleus assay in human lymphocytes. In the 28 day and 90 day repeated dose toxicity studies no test item related mortality or toxicological effects on clinical signs, body weight, food consumption, urine parameters, hematology, clinical biochemistry parameters, organ weight, gross pathological findings and histopathology were observed. Based on the studies described here, MYOLUTION® does not exert a genotoxic effect, and a no-observed-adverse-effect-level of 3318.38 mg/kg bw/day in males and 3733.28 mg/kg bw/day in females was determined in the 90 day repeated dose toxicity study.