Chinese hamster ovary cell assays for mutation and chromosome damage: data from non-carcinogens

In vitro genotoxicity tests are employed to screen chemicals for their capability to cause various DNA and chromosomal alterations, and the results are often used to predict their potential for carcinogenicity. However, there is controversy regarding the apparent low specificity of some in vitro genotoxicity assays, which result in a high false positive rate. Since we use and rely upon in vitro assays for risk assessment and prediction of carcinogenicity, this specificity issue is of serious concern to us. Hence, we selected ten compounds deemed non-carcinogens in the literature to test for the induction of gene mutation and chromosomal damage using the Chinese hamster ovary cell/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) mutation assay performed concurrently with a CHO micronucleus assay. The chemical exposures for the two end-points were done simultaneously. The protocol for the two end-points was developed using the carcinogens N-methyl-N'-nitro-N-nitrosoguanidine, 3-methylcholanthrene, cyclophosphamide and 7,12-dimethylbenzanthracene. The non-carcinogens chosen were 4-nitro-o-phenylenediamine, p-phenylenediamine dihydrochloride, 3-nitropropionic acid, dichlorvos, 2-(chloromethyl)pyridine, N-(1-naphthyl)ethylenediamine 2HCl, O-anthranilic acid, 4-nitroanthranilic acid, anilazine and triphenyltin hydroxide. Each of these chemicals had been reported positive in the Ames test and/or the mouse lymphoma TK+/- mutation assay. In addition, eight of them were also reported positive in in vitro assays for chromosome aberrations and/or sister chromatid exchange (SCE). We found four of the ten chemicals negative for gene mutation and micronucleus induction without and with activation in the CHO/HGPRT mutation and CHO micronucleus assays. However, one of these four chemicals may be a potential carcinogen according to other carcinogenicity reviewers. Four other chemicals that induced only micronuclei were negative for gene mutation. Dichlorvos was positive for gene mutation and micronucleus induction without and with activation. This chemical has been shown recently to cause various tumors in rodents. One of the non-carcinogens was positive in the micronucleus test and equivocally positive in the mutation test. These results indicate that the CHO/HGPRT mutation assay may provide more relevant results than the CHO micronucleus assay, the mouse lymphoma mutation assay, or in vitro SCE and chromosome aberration assays when screening chemicals for potential carcinogenicity.     

Evaluation of genotoxicity of nitrile fragrance ingredients using in vitro and in vivo assays

Genotoxicity studies were conducted on a group of 8 fragrance ingredients that belong to the nitrile family. These nitriles are widely used in consumer products however there is very limited data in the literature regarding the genotoxicity of these nitriles. The 8 nitriles were assessed for genotoxicity using an Ames test, in vitro chromosome aberration test or in vitro micronucleus test. The positive results observed in the in vitro tests were further investigated using an in vivo micronucleus test. The results from these different tests were compared and these 8 nitriles are not considered to be genotoxic. Dodecanitrile and 2,2,3-trimethylcyclopent-3-enylacetonitrile were negative in the in vitro chromosome aberration test and in vitro micronucleus test, respectively. While citronellyl nitrile, 3-methyl-5-phenylpentanenitrile, cinnamyl nitrile, and 3-methyl-5-phenylpent-2-enenitrile revealed positive results in the in vitro tests, but confirmatory in vivo tests determined these nitriles to be negative in the in vivo micronucleus assay. The remaining two nitriles (benzonitrile and α-cyclohexylidene benzeneacetonitrile) were negative in the in vivo micronucleus test. This study aims to evaluate the genotoxicity potential of these nitriles as well as enrich the literature with genotoxicity data on fragrance ingredients.     

Selection and Evaluation of an Appropriate Model for Screening Adenosine Analogs for Chromosome Aberrations

As part of a preclinical safety assessment profile, the adenosine analog SC-46267, a candidate antihypertensive drug, was tested for potential genotoxicity in a series of in vitro and in vivo assays. Negative results were obtained in the Salmonella/microsome and CHO/HGPRT point mutation assays and in an acute mouse micronucleus test. However, in the presence of exogenous metabolism, positive results in the mouse lymphoma L5178Y assay and an in vitro chromosome aberration assay with purified rat lymphocytes indicated that the analog was a potential clastogen. Both adenosine and the analog were subsequently tested in rat lymphocytes using whole-blood versus purified lymphocyte cultures. High concentrations of adenosine and the adenosine analog induced chromosome aberrations in the purified lymphocytes. However, neither compound induced chromosome damage when cultured in the presence of whole blood, regardless of the presence or absence of the metabolic activation system. These results were similar to those reported in the literature that suggested that high concentrations of adenosine and other naturally occurring nucleotides can produce nonphysiological perturbations of nucleotide pools that are clastogenic in purified cultures of lymphocytes but not in whole-blood cultures. Our studies appear to support the hypothesis that adenosine deaminase, present at normal physiological levels in whole blood, is capable of detoxifying excessive levels of adenosine. Both compounds were also negative in a 10-day repeated dose mouse micronucleus test, which further ensures that the positive in vitro findings are not biologically significant. An in vitro clastogenicity assay using whole blood appears to be more appropriate than use of purified lymphocytes for screening adenosine analogs. The results from this series of experiments demonstrate the importance of considering culture conditions when designing in vitro test protocols. Furthermore, this study exemplifies the need to explore the mechanisms of activity as well as in vivo relevance of in vitro results.     

国产环丙沙星的诱变性和致癌性研究

应用小鼠骨髓细胞微核试验、CHL细胞染色体畸变试验及叙利亚地鼠胚胎细胞转化试验,对国产环丙沙星进行了诱变性和致癌研究。结果: 环丙沙星在小鼠骨髓细胞微核试验和CHL细胞染色体畸变试验中均为阴性;在细胞转化试验中不诱发SHE细胞发生形态转化。实验结果提示环丙沙星在体内、外不引起染色体损伤;无体外致癌作用。     

聚乙二醇修饰降纤酶的遗传毒性评价

目的检测聚乙二醇修饰降纤酶的遗传毒性。方法应用鼠伤寒沙门菌回复突变试验(Ames试验)、体外培养CHO细胞染色体畸变试验和小鼠骨髓微核试验检测聚乙二醇修饰降纤酶的遗传毒性。结果 Ames试验结果显示每平皿100、20、4、0.8、0.16 U各个剂量组,在加或不加S9代谢活化系统时对组氨酸缺陷型鼠伤寒沙门菌TA97、TA98、TA100、TA102及TA1535所诱发的回复突变菌落数均与溶剂对照的突变菌落数相近。体外培养CHO细胞染色体畸变试验结果显示2.5、5.0和10.0 U.mL-1各个剂量组在加S9代谢活化系统于24 h和不加S9代谢活化系统于24 h、48 h培养的CHO细胞染色体畸变率与溶剂对照组比较均无显著差异(P>0.05)。小鼠骨髓微核试验显示425、850、1700 U.kg-1各个剂量组对ICR小鼠的微核诱发率与溶剂对照组比较均无显著差异(P>0.05)。结论聚乙二醇修饰降纤酶对鼠伤寒沙门菌无致突变性,对哺乳动物培养细胞的染色体无致畸变作用,对ICR小鼠无诱发骨髓嗜多染红细胞微核的效应。表明聚乙二醇修饰降纤酶在本实验条件下无遗传毒性。     

The comet assay with multiple mouse organs: comparison of comet assay results and carcinogenicity with 208 chemicals selected from the IARC monographs and U.S. NTP Carcinogenicity Database

The comet assay is a microgel electrophoresis technique for detecting DNA damage at the level of the single cell. When this technique is applied to detect genotoxicity in experimental animals, the most important advantage is that DNA lesions can be measured in any organ, regardless of the extent of mitotic activity. The purpose of this article is to summarize the in vivo genotoxicity in eight organs of the mouse of 208 chemicals selected from International Agency for Research on Cancer (IARC) Groups 1, 2A, 2B, 3, and 4, and from the U.S. National Toxicology Program (NTP) Carcinogenicity Database, and to discuss the utility of the comet assay in genetic toxicology. Alkylating agents, amides, aromatic amines, azo compounds, cyclic nitro compounds, hydrazines, halides having reactive halogens, and polycyclic aromatic hydrocarbons were chemicals showing high positive effects in this assay. The responses detected reflected the ability of this assay to detect the fragmentation of DNA molecules produced by DNA single strand breaks induced chemically and those derived from alkali-labile sites developed from alkylated bases and bulky base adducts. The mouse or rat organs exhibiting increased levels of DNA damage were not necessarily the target organs for carcinogenicity. It was rare, in contrast, for the target organs not to show DNA damage. Therefore, organ-specific genotoxicity was necessary but not sufficient for the prediction of organ-specific carcinogenicity. It would be expected that DNA crosslinkers would be difficult to detect by this assay, because of the resulting inhibition of DNA unwinding. The proportion of 10 DNA crosslinkers that was positive, however, was high in the gastrointestinal mucosa, stomach, and colon, but less than 50% in the liver and lung. It was interesting that the genotoxicity of DNA crosslinkers could be detected in the gastrointestinal organs even though the agents were administered intraperitoneally. Chemical carcinogens can be classified as genotoxic (Ames test-positive) and putative nongenotoxic (Ames test-negative) carcinogens. The Ames test is generally used as a first screening method to assess chemical genotoxicity and has provided extensive information on DNA reactivity. Out of 208 chemicals studied, 117 are Ames test-positive rodent carcinogens, 43 are Ames test-negative rodent carcinogens, and 30 are rodent noncarcinogens (which include both Ames test-positive and negative noncarcinogens). High positive response ratio (110/117) for rodent genotoxic carcinogens and a high negative response ratio (6/30) for rodent noncarcinogens were shown in the comet assay. For Ames test-negative rodent carcinogens, less than 50% were positive in the comet assay, suggesting that the assay, which detects DNA lesions, is not suitable for identifying nongenotoxic carcinogens. In the safety evaluation of chemicals, it is important to demonstrate that Ames test-positive agents are not genotoxic in vivo. This assay had a high positive response ratio for rodent genotoxic carcinogens and a high negative response ratio for rodent genotoxic noncarcinogens, suggesting that the comet assay can be used to evaluate the in vivo genotoxicity of in vitro genotoxic chemicals. For chemicals whose in vivo genotoxicity has been tested in multiple organs by the comet assay, published data are summarized with unpublished data and compared with relevant genotoxicity and carcinogenicity data. Because it is clear that no single test is capable of detecting all relevant genotoxic agents, the usual approach should be to carry out a battery of in vitro and in vivo tests for genotoxicity. The conventional micronucleus test in the hematopoietic system is a simple method to assess in vivo clastogenicity of chemicals. Its performance is related to whether a chemical reaches the hematopoietic system. Among 208 chemicals studied (including 165 rodent carcinogens), 54 rodents carcinogens do not induce micronuclei in mouse hematopoietic system despite the positive finding with one or two in vitro tests. Forty-nine of 54 rodent carcinogens that do not induce micronuclei were positive in the comet assay, suggesting that the comet assay can be used as a further in vivo test apart from the cytogenetic assays in hematopoietic cells. In this review, we provide one recommendation for the in vivo comet assay protocol based on our own data.     

GADD45a-GFP GreenScreen HC genotoxicity screening assay

BACKGROUND: Genetic toxicology is getting very interesting. The International Conference on Harmonisation has drafted new guidance that allows for the registration of pharmaceuticals without the submission of data from in vitro mammalian genotoxicity tests (in vitro micronucleus test, chromosomal aberrations, mouse lymphoma assay). These tests often produce falsely positive predictions of genotoxic carcinogenicity. OBJECTIVES: This article reviews the properties of the Gadd45a-GFP (green fluorescent protein) assay, for which positive results appear to provide more reliable predictions of genotoxic carcinogenicity. The criteria for assessment of genotoxicity assays are reviewed. Consideration is given to the value of genotoxicity hazard assessment early in pharmaceutical discovery. METHODS: Peer-reviewed data have been reviewed, as well as information contributed to the public domain through conference presentations. RESULTS/CONCLUSION: The Gadd45a assay is increasingly used as a screening tool, and has utility in the prioritisation of Ames-negative compounds prior to in vivo genotoxicity assessment.     

Genotoxicity of motorcycle exhaust particles in vivo and in vitro.

We studied the genotoxic potency of motorcycle exhaust particles (MEP) by using a bacterial reversion assay and chromosome aberration and micronucleus tests. In the bacterial reversion assay (Ames test), MEP concentration-dependently increased TA98, TA100, and TA102 revertants in the presence of metabolic-activating enzymes. In the chromosome aberration test, MEP concentration-dependently increased abnormal structural chromosomes in CHO-K1 cells both with and without S9. Pretreatment with antioxidants (alpha-tocopherol, ascorbate, catalase, and NAC) showed varying degrees of inhibitory effect on the MEP-induced mutagenic effect and chromosome structural abnormalities. In the in vivo micronucleus test, MEP dose-dependently induced micronucleus formation in peripheral red blood cells after 24 and 48 h of treatment. The increase of micronucleated reticulocytes induced by MEP was inhibited by pretreatment with alpha-tocopherol and ascorbate. The fluorescence intensity of DCFH-DA-loaded CHO-K1 cells was increased upon the addition of MEP. Our data suggest that MEP can induce genotoxicity through a reactive oxygen species-(ROS-) dependent pathway, which can be augmented by metabolic activation. Alpha-tocopherol, ascorbate, catalase, and NAC can inhibit MEP-induced genotoxicity, indicating that ROS might be involved in this effect.     

Use of the alkaline comet assay for industrial genotoxicity screening: comparative investigation with the micronucleus test.

We evaluated the suitability of the alkaline comet assay as a screening test in industrial routine testing of new chemicals. Thirty-six pharmaceutical compounds with unknown genotoxic potential were tested comparatively in the comet assay and micronucleus test (MNT) using V79 Chinese hamster cells. The comparison of results is generally based on at least two independent experiments, each with two replicate cultures at a minimum of three concentrations. We found a high degree of concordance between results of the comet assay and MNT. All compounds with negative MNT results were also negative in the comet assay. All positive compounds in the comet assay were also positive in the MNT. However, 16 of 38 positive MNT results were negative in the comet assay. Some of the contrary findings may be due to aneugenic effects, which are detected in the MNT but not in the comet assay. However, the majority of the contrary results may be a consequence of cytotoxicity, which can induce elevated micronucleus frequencies but may not lead to positive effects in the comet assay. Additional data of 39 compounds tested in the Ames test and the comet assay were compared. Four of these compounds that were Ames positive were also positive in the comet assay. However, the comet assay also detected 16 compounds that were negative in the Ames test. We believe that the comet assay in vitro is a useful, fast screening system in mammalian cells that can be used in a test battery during drug development.     

Appropriate in vitro test conditions for genotoxicity testing of fibers

With the exception of asbestos fibers, little information is available on genotoxicity testing of fibers (i.e., respirable-sized, fiber-shaped particulates, RFP). In contrast to standard genotoxicity testing of soluble substances, fibers bring about specific problems. Test results can be influenced by fiber dimensions, surface properties, and biopersistence. The mechanisms of fiber-induced genotoxicity are not yet clear, but direct interaction with the genetic material and indirect effects via production of reactive oxygen species (ROS) have been proposed. Asbestos did not significantly induce gene mutations in bacterial and mammalian systems but led to a clear induction of structural and numerical chromosome aberrations in cultured mammalian cells. It is the purpose of this article to critically review positive in vitro genotoxicity data obtained for asbestos in the comet assay, the chromosome aberration test, and the micronucleus test and to identify the test conditions that are specifically required for the detection of asbestos-induced genotoxicity. It is concluded that appropriate cell systems are available for testing fiber-induced genotoxicity. Fiber samples have to be well characterized and phagocytosis and cytotoxic effects have to be determined for the correct interpretation of genotoxicity test results. A combination of the micronucleus test and the comet assay using continuous treatment (without exogenous metabolic activation) seems to be well suited to detect genotoxic activity of asbestos fibers with high accuracy. Further investigations are needed to shown whether this approach can be recommended for genotoxicity testing of fibers in general.     

Assessment of the genotoxic potential of Caramel Colour I in four short-term tests.

A battery of three short-term tests in vitro and one in vivo was used to determine the genotoxicity of Caramel Colour I. The results of the bacterial mutation assay, using five strains of Salmonella typhimurium, and the mouse micronucleus assay in vivo showed no evidence of genotoxic activity. Results from both the cytogenetics assay in vitro, using CHO cells, and the mouse lymphoma assay indicated that there was some genotoxic activity associated with Caramel Colour I but only in the absence of S-9 and at very high dose levels.     

白障明的诱变性试验研究

作者用鼠伤寒沙门氏菌回变试验,小鼠骨髓红细胞微核试验及CHL细胞染色体畸变试验,对白障明进行了诱变性试验研究。结果表明鼠伤寒沙门氏菌回变试验为阴性,该药可能不诱发基因突变,NIH小鼠骨髓红细胞微核试验及CHL细胞染色体畸变试验均为阴性,说明该药可能不诱发体内,体外染色体损伤作用。     

The absence of genotoxicity of sucralose

Sucralose is a non-nutritive sweetener that is approximately 600 times sweeter than table sugar. It is currently approved for use in over 80 countries. Evidence from chronic studies demonstrates that this compound is not carcinogenic. This report summarizes the results of genotoxicity studies that were part of the original safety assessment of sucralose–conducted early in the safety investigation and shared with regulatory agencies around the world. Studies included the Ames (Salmonella typhimurium) reverse mutation test, the Escherichia coli pol A+/A− test, an in vitro chromosome damage assay in human lymphocytes, mutation in TK +/− mouse lymphoma cells, an in vivo chromosome aberration test in rats and two separate micronucleus tests in mice. All results were evaluated as negative. These results support the overall conclusion by regulatory and heath agencies that sucralose is safe for its intended use.     

Evaluation of 5-aminoisoquinoline (5-AIQ), a novel PARP-1 inhibitor for genotoxicity potential in vitro and in vivo

5-aminoisoquinoline (5-AIQ) is an active PARP-1 inhibitor as well as an important functional group various drugs. Quinolines are generally known as mutagenic and carcinogenic in various in vitro and in vivo systems, while both positive and negative findings are available on the mutagenic potential of several isoquinolines. Since no literature is available on the genotoxicity of 5-AIQ, a battery of tests were conducted, in accordance with relevant OECD protocols, such as bacterial reverse mutation test, in vitro chromosomal aberration test, and bone marrow micronucleus test in mouse. These studies demonstrate that 5-AIQ does not possess genotoxic activity both with in vitro and in vivo systems. The findings substantiate the therapeutic value of 5-AIQ.     

Genotoxicity studies on selected organosilicon compounds: in vitro assays

A series of 12 organosilicon compounds representing potential intermediates in the synthesis and degradation of polydimethylsiloxanes were evaluated for genotoxic potential with a battery of in vitro assays. Microbial assays included the Ames bacterial reverse mutation in Salmonella, mitotic gene conversion in Saccharomyces cerevisiae D4 and DNA repair in E. Coli pol A +/-. These assays were conducted with and without a metabolic activation system containing Aroclor 1254-induced rat-liver homogenate. Forward gene mutation, sister-chromatid exchange, DNA alkaline elution and chromosome aberration potential were evaluated in mouse lymphoma L5178Y tissue culture cells. The tissue culture assays were performed with and without metabolic activation mixture utilizing uninduced mouse-liver S-9. The use of this enzyme preparation was felt to more closely mimic the actual in vivo situation and to be more compatible with mouse cells employed in the assay. No evidence of gene mutation was observed. However, six of the 12 compounds evaluated demonstrated potential in vitro clastogenic (chromosome damaging) activity.     

Evaluation of the GADD45α‐GFP GreenScreen HC assay for rapid and reliable in vitro early genotoxicity screening

Twenty‐two of Galderma's proprietary compounds were tested in the GADD45α‐GFP ‘GreenScreen HC’ assay (GS), the SOS‐ChromoTest and the Mini‐Ames to evaluate GSs performance for early genotoxicity screening purposes. Forty more characterized compounds were also tested, including antibiotics: metronidazole, clindamycin, tetracycline, lymecycline and neomycin; and catecholamines: resorcinol mequinol, hydroquinone, one aneugen carbendazim, one corticoid dexamethasone, one peroxisome proliferator‐activated receptor rosiglitazone, one pesticide carbaryl and two further proprietary molecules with in vitro genotoxicity data. With proprietary molecules, this study concluded that the GS renders the SOS‐ChromoTest obsolete for in vitro screening. The GS confirmed all results of the Mini‐Ames test (100% concordance). Compared with the micronucleus test, the GS showed a concordance of 82%. With known compounds, the GS ranked the potency of positive results for catecholamines in accordance with other genotoxicity tests and showed very reproducible results. It confirmed positive results for carbendazim, for tetracycline antibiotics and for carbaryl. The GS produced negative results for metronidazole, a nitroreduction‐specific bacterial mutagen, for dexamethasone (a non‐genotoxic apoptosis inducer), for rosiglitazone (a GADD45γ promoter inducer) and for clindamycin and neomycin (inhibitors of macromolecular synthesis in bacteria). As such, the GS appears to be a reproducible, robust, specific and sensitive test for genotoxicity screening. Copyright © 2012 John Wiley & Sons, Ltd.     

Genotoxicity study of bojungchisup-tang, an oriental herbal decoction-in vitro chromosome aberration assay in Chinese hamster lung cells andin vivo supravital-staining micronucleus assay with mouse peripheral reticulocytes

The toxicity evaluation of oriental herbal drugs is of great concern at present. Bojungchisuptang (BCST, in Korean), a decocted medicine of oriental herbal mixture, is now well used in clinic at oriental hospitals for the treatment of edema of several diseases in practice. However, the toxicity of the oriental herbal decocted medicines such as genetic toxicity is not well defined until now. In this respect, to clarify the genetic toxicity of BCST,in vitro chromosome aberration assay with Chinese hamster lung (CHL) fibroblasts andin vivo supravital micronucleus assay with mouse peripheral reticulocytes were performed in this study. In the chromosome aberration assay, we used 5,000 μg/ml BCST as maximum concentration because no remarkable cytotoxicity in CHL cells was observed both in the presence and absence of S-9 metabolic activation system. No statistical significant differences of chromosome aberrations were observed in CHL cells treated with 5,000, 2,500 and 1,250 μg/ml BCST for 6 hour both in the presence and absence of S-9 metabolic activation. However, very weak positive result (6.5∼8.0% aberration) of BCST was obtained in the absence of S-9 metabolic activation system at 5,000 μg/ml BCST when treated for 24 hour, i.e. 1.5 normal cell cycle time. And also,in vivo clastogenicity of BCST was studied by acridine orange-supravital staining micronucleus assay using mouse peripheral reticulocytes. We used 2,000 mg/kg as the highest oral dose in this micronucleus assay because no acute oral toxicity of BCST was observed in mice. The optimum induction time of micronucleated reticulocytes (MNRETs) was determined as 36 hours after oral administration of 2,000 mg/kg BCST. No significant differences of MNRETs between control and BCST treatment groups were observedin vivo micronucleus assay. From these results, BCST revealed very weak positive result in chromosome aberration assayin vitro with CHL cells and no clastogenicity in micronucleus assayin vivo.     

Size- and coating-dependent cytotoxicity and genotoxicity of silver nanoparticles evaluated using in vitro standard assays

The physicochemical characteristics of silver nanoparticles (AgNPs) may greatly alter their toxicological potential. To explore the effects of size and coating on the cytotoxicity and genotoxicity of AgNPs, six different types of AgNPs, having three different sizes and two different coatings, were investigated using the Ames test, mouse lymphoma assay (MLA) and in vitro micronucleus assay. The genotoxicities of silver acetate and silver nitrate were evaluated to compare the genotoxicity of nanosilver to that of ionic silver. The Ames test produced inconclusive results for all types of the silver materials due to the high toxicity of silver to the test bacteria and the lack of entry of the nanoparticles into the cells. Treatment of L5718Y cells with AgNPs and ionic silver resulted in concentration-dependent cytotoxicity, mutagenicity in the Tk gene and the induction of micronuclei from exposure to nearly every type of the silver materials. Treatment of TK6 cells with these silver materials also resulted in concentration-dependent cytotoxicity and significantly increased micronucleus frequency. With both the MLA and micronucleus assays, the smaller the AgNPs, the greater the cytotoxicity and genotoxicity. The coatings had less effect on the relative genotoxicity of AgNPs than the particle size. Loss of heterozygosity analysis of the induced Tk mutants indicated that the types of mutations induced by AgNPs were different from those of ionic silver. These results suggest that AgNPs induce cytotoxicity and genotoxicity in a size- and coating-dependent manner. Furthermore, while the MLA and in vitro micronucleus assay (in both types of cells) are useful to quantitatively measure the genotoxic potencies of AgNPs, the Ames test cannot.     

The genotoxicity of diaveridine and trimethoprim

We examined the genotoxicity of diaveridine and trimethoprim in the bacterial umu test, the bacterial reverse mutation test, the in vitro chromosome aberration test, the in vivo rodent bone marrow micronucleus test in two species, and the in vivo comet assay in five mouse organs. Both compounds were negative in the umu test (Salmonella typhimurium TA1535/pSK1002) and in the reverse mutation tests (S. typhimurium TA100, TA98, TA97, TA102, and Escherichia coli WP2 uvrA/pKM101) in the presence and absence of S9 mix. Diaveridine induced structural chromosome aberrations in cultured Chinese hamster CHL cells in the absence of a metabolic activation system, but not in the presence of a liver S9 fraction. No clastogenic activity in CHL cells was detected for trimethoprim. Bone marrow micronucleus tests in mice and rats conducted on diaveridine by single- and triple-oral dosing protocols were negative. The comet assay revealed that a single oral administration of diaveridine significantly induced DNA damage in liver, kidney, lung, and spleen cells, but not in bone marrow cells. The significant increase in migration values of DNA was reproducible with dose-response relationship. We suggest that the liver detoxifies the compound before it reaches the bone marrow, and that is why it is negative in the in vivo bone marrow micronucleus test. We concluded that diaveridine is genotoxic to mammalian cells in vitro and in vivo.     

An assessment of the genotoxicity and human health risk of topical use of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one].

Kojic acid (KA), a natural substance produced by fungi or bacteria, such as Aspergillus, Penicillium or Acetobacter spp, is contained in traditional Japanese fermented foods and is used as a dermatological skin-lightening agent. High concentrations of KA (>or=1000 microg/plate) were mutagenic in S. typhimurium strains TA 98, TA 100, TA 1535, TA102 and E. coli WP2uvrA, but not in TA 1537. An Ames test following the "treat and plate" protocol was negative. A chromosome aberration test in V79 cells following a robust protocol showed only a marginal increase in chromosome aberrations at cytotoxic concentrations after prolonged (>or=18 h) exposure. No genotoxic activity was observed for hprt mutations either in mouse lymphoma or V79 cells, or in in vitro micronucleus tests in human keratinocytes or hepatocytes. All in vivo genotoxicity studies on KA doses were negative, including mouse bone marrow micronucleus tests after single or multiple doses, an in vivo/in vitro unscheduled DNA synthesis (UDS) test, or a study in the liver of the transgenic Muta(TM) Mouse. On the basis of pharmacokinetic studies in rats and in vitro absorption studies in human skin, the systemic exposure of KA in man following its topical application is estimated to be in the range of 0.03-0.06 mg/kg/day. Comparing these values with the NOAEL in oral subchronic animal studies (250 mg/kg/day), the calculated margin of safety would be 4200- to 8900-fold. Comparing human exposure with the doses that were negative for micronuclei, UDS and gene mutations in vivo, the margins of safety are 16000 to 26000-fold. In conclusion, the topical use of KA as a skin lightening agent results in minimal exposure that poses no or negligible risk of genotoxicity or toxicity to the consumer.