GRAS Determination Scientific Procedures and Possible Alternatives

The use of a food substance is Generally Recognized as Safe (GRAS) through scientific procedures or experience based on common use in food. The pivotal data used for GRAS determination must be of common knowledge and should include evidence for safety under the conditions of intended use of the substance. Such evidence includes data on the identity and specifications of the substance, its properties of absorption, distribution, metabolism and excretion, and depending on the level of concern, data on genotoxicity, acute and subchronic toxicity, reproductive and developmental toxicity and carcinogenicity. Several alternative procedures can be used as the replacement for standard scientific procedures in order to improve the GRAS process

Testing of Flavor and Fragrance Materials in Turkey Egg Genotoxicity Assay (TEGA) and Comparison of the Results in Ovo, in Vitro and in Vivo

The aim of the study was to investigate the use of the Turkey Egg Genotoxicity Assay (TEGA) as a non-animal alternative to in vivo follow-up studies. The genotoxic potential of 19 diverse flavor and fragrance (F&F) agents was assessed in the TEGA using 32P-nucleotide postlabeling (NPL) and comet assays to detect hepatic DNA adducts and strand breaks, respectively. The compounds were selected for testing based on their chemical structures and results in the GADD45a-Gluc ‘BlueScreen HC’ (BSHC) genotoxicity and the Ames mutagenicity assays. Two F&F materials (BDHCA and MEU) produced DNA adducts, and four materials (BDHCA, BMHCA, HEX and MAL) produced DNA strand breaks in the NPL and comet assays, respectively. Fourteen other tested compounds were negative in both NPL and comet assays. Of these14 materials, only 3 are negative in vitro, yet the majority of these materials are not shown to be genotoxic. Based on reports of oxidative DNA damage for two of the materials (MAL & HDMF), these compounds were tested in the enhanced comet assay using repair enzymes to identify oxidative DNA damage. In the enhanced comet assay positive comet findings for MAL were not confirmed, and only equivocal evidence of oxidative damage was found. Meanwhile, HDMF, produced positive results in the enhanced comet assay with formamidopyrimidine DNA glycosylase (FPG) enzyme digestion. In the test set, the TEGA had better specificity and sensitivity for in vivo data compared to in vitro test results. The observed non-concordance with in vitro data could be due to differences in the endpoints measured by the TEGA compared to those in vitro or due to the acknowledged higher rate of false positive results in in vitro systems. The TEGA showed high accuracy compared to standard in vivo genotoxicity assays for the prediction of genotoxic carcinogens as positive and the non-(genotoxic) carcinogens as negative. This findings are of high importance, since the end result of standard genotoxicity assays is typically the prediction of cancer risk

DNA-Damaging Activities of Twenty-Four Structurally Diverse Unsubstituted and Substituted Cyclic Compounds in Embryo-Fetal Chicken Livers

DNA-damaging activities of twenty-four structurally diverse unsubstituted and substituted cyclic compounds were assessed in embryo-fetal chicken livers. Formation of DNA adducts and strand breaks were measured using the nucleotide (32)P-postlabelling (NPL) and comet assays, respectively. Unsubstituted monocyclic benzene, polycyclic fused ring compound naphthalene, covalently connected polycyclic ring compound biphenyl, and heterocyclic ring compound fluorene did not produce DNA damage. Amino-substituted monocyclic compounds, aniline and p-phenylenediamine, as well as polycyclic 1-naphthylamine were also negative. In contrast, carcinogenic monocyclic methyl-substituted anilines: o-toluidine, 2,6-xylidine, 3,4-dimethylaniline, 4-chloro-o-toluidine; 2 methoxy-substituted methylaniline: p-cresidine; 2,4 and 2,6 diamino- or dinitro- substituted toluenes all produced DNA damage. Genotoxic polycyclic amino-substituted 2-naphthylamine, 4-aminobiphenyl, benzidine, methyl-substituted 3,2\u27-dimethyl-4-aminobiphenyl and 4-dimethylaminoazobenzene as well as amino- and nitro- fluorenes substituted at the 1 or 2 positions also were positive in at least one of the assays. Overall, the DNA damaging activity of cyclic compounds in embryo-fetal chicken livers reflected the type and position of the substitution on the aromatic ring. Additionally, substituted polycyclic compounds exhibited higher DNA-damaging potency compared to monocyclic chemicals. These results are congruent with in vivo findings in other species, establishing chicken eggs as a reliable system for structure-activity assessment of members of groups of related chemicals

Structure-Activity Relationships for DNA Damage by Alkenylbenzenes in Turkey Egg Fetal Liver

Certain alkenylbenzenes (AB), flavoring chemicals naturally occurring in spices and herbs, are established to be cytotoxic and hepatocarcinogenic in rodents. The purpose of the present study was to determine the DNA damaging potential of key representatives of this class using the Turkey Egg Genotoxicity Assay. Medium white turkey eggs with 22- to 24-day-old fetuses received three injections of nine AB with different carcinogenic potentials: safrole (1, 2 mg/egg), methyl eugenol (2, 4 mg/egg), estragole (20, 40 mg/egg), myristicin (25, 50 mg/egg), elemicin (20, 50 mg/egg), anethole (5, 10 mg/egg), methyl isoeugenol (40, 80 mg/egg), eugenol (1, 2.5 mg/egg), and isoeugenol (1, 4 mg/egg). Three hours after the last injection, fetal livers were harvested for measurement of DNA strand breaks, using the comet assay and DNA adducts formation, using the nucleotide(3) (2)P-postlabeling assay. Estragole, myristicin, and elemicin induced DNA stand breaks. These compounds as well as safrole, methyl eugenol and anethole, at the highest doses tested, induced DNA adduct formation. Methyl isoeugenol, eugenol, and isoeugenol did not induce genotoxicity. The genotoxic AB all had the structural features of either a double bond in the alkenyl side chain at the terminal 2\u27,3\u27-position, favorable to formation of proximate carcinogenic 1\u27-hydroxymetabolite or terminal epoxide, or the absence of a free phenolic hydroxyl group crucial for formation of a nontoxic glucuronide conjugate. In contrast, methyl isoeugenol, eugenol and isoeugenol, which were nongenotoxic, possessed chemical features, unfavorable to activation

Histologic and Genomic Evaluation of Liver Cell Proliferation in the Chicken Egg Genotoxicity Assay (CEGA)

A variety of genotoxic carcinogens tested in the Chicken Egg Genotoxicity Assay (CEGA), which assesses liver DNA strand breaks and adducts, also interfered with fetal liver proliferation, differentiation and migration, leading to hepatocellular dysplasia and distortion of trabecular pattern. The present study assesses cell proliferation and gene expression profile in fetal chicken livers, since these processes are involved in embryo-fetal development and their dysregulation can lead to neoplastic development. Groups of at least 12 white leghorn chicken eggs were administered the vehicles for CEGA, deionized water (DW) and 20% aqueous solution of Solutol HS15 (20% HS15), in 3 daily injections on days 9 - 11 of incubation. The group used as control did not receive any injections. Three hours after the last injection, half of the livers were collected for gene expression analysis. One day (day 12 of incubation) and one week (day 18) after dosing was discontinued, the remaining livers were collected for histologic analysis. At day 12, in sections stained with hematoxylin and eosin (H&E), in all groups, nascent trabecular hepatocellular pattern was established and normal liver cells and extracellular matrix elements were present. At day 18, the hepatocellular trabecular pattern was completed. Immunohistochemical staining for proliferating cell nuclear antigen (PCNA) showed no significant differences in cell proliferation between the groups, the percentage of proliferating cells at 12 and 18 days was in the range of 61% to 65%. Thus, cellular proliferation in fetal chicken livers was not affected by DW and HS 15. Gene expression analysis using chicken 44K Agilent microarray, revealed that DW had minimal effect on the expression of genes involved in the regulation of cell cycle and proliferation, while downregulation of two tumor suppressor genes p53 and APC was observed. HS15 significantly deregulated multiple genes involved in cell cycle and proliferation pathways. Among genes upregulated were Rad21, p300, Mdm2, Mad1. Downregulated genes included APC, CycA, CycB, TGFβ, p53, GADD45 and PCNA. Despite alterations in the expression of genes involved in cell cycle and proliferation produced by HS15, fetal chicken hepatocytes evidently maintained their proliferative status. A 30% aqueous solution of ethanol (30% ETOH) was tested in CEGA for its effects on gene expression as a possible substitution for 20% HS15. Similar to DW, 30% ETOH did not cause significant alterations in gene expression profile. Thus, it can be used as a vehicle in CEGA

In ovo Testing of Flavor and Fragrance Materials in Turkey Egg Genotoxicity Assay (TEGA), Comparison of Results to in Vitro and in Vivo Data

Genotoxicity of flavor and fragrance materials was assessed in Turkey Egg Genotoxicity Assay (TEGA) using (32)P-nucleotide postlabeling (NPL) and comet assays to detect hepatic DNA adducts and strand breaks. Twenty materials having results in GADD45a-Gluc \u27BlueScreen HC\u27 genotoxicity assay, and standard in vitro and in vivo tests, were selected to evaluate the accuracy of TEGA. Quinoline (QUI) and 2-acetylaminofluorene (AAF) served as positive comparators. Two materials, p-tert-butyldihydrocinnamaldehyde (BDHCA) and methyl eugenol (MEU) produced DNA adducts. BDHCA, p-t-butyl-alpha-methylhydrocinnamic aldehyde (BMHCA), trans-2-hexenal (HEX) and maltol (MAL) produced DNA strand breaks. Fifteen other materials were negative in both assays. Based on reports of oxidative DNA damage induction by MAL and 4-hydroxy-2.5-dimethyl-3(2H) furanone (HDMF), modified comet assays were conducted. Positive comet findings for MAL were not confirmed, and only equivocal evidence of oxidative damage was found. Accordingly, MAL was judged to have equivocal genotoxicity in TEGA. HDMF was positive in modified comet assay, indicating an ability to produce oxidative DNA damage. TEGA showed modest concordance with results in regulatory in vitro assays. Findings in TEGA, with few exceptions, were concordant with the results of in vivo genotoxicity and carcinogenicity testing. Thus, TEGA is an attractive alternative model for the assessment of genotoxic potential of chemicals in vivo