Genotoxicity and acute toxicity evaluation of the three amino acid additives with Corynebacterium glutamicum biomass

Highlights • The three amino acid additives with C. glutamicum were found to be non-mutagenic and non-toxic.• Genotoxicity is not observed in Ames assay and mammalian cytogenentic assay of the three amino acid additives.• No clinical signs, mortalities, abnormal necropsy findings were observed in acute oral toxicity in both Sprague-Dawley and Wistar rats.


Introduction
Dietary ingestion of amino acids in domestic animals has been widely investigated and the important role of amino acids in modulation of metabolism, growth, reproduction and milk production was unveiled [1]. Accordingly, the addition of amino acids in animal feed is a reasonable option for livestock and the animal breeding industry. For instance, L-tryptophan is one of the essential amino acids in mammals which cannot be synthesized. Diverse roles of L-tryptophan were identified such as food intake enhancer, growth contributor, and immune response modulator [2]. L-threonine is one of the first-limiting amino acids in low protein diet of animals and supplementation of threonine was reported to improve lipid metabolism in poultry and to increase whole-body protein synthesis in weanling colts [3,4]. Deprivation of branched chain amino acid L-valine was known to remarkably decrease insulin sensitivity of mice and impair immune system of young grass carp [5,6]. On the contrary, optimal dietary supplementation of valine increases body weight gain of Pekin duck [7].
Corynebacterium glutamicum (C. glutamicum) is a gram-positive and non-pathogenic bacterium, which occurs naturally in soil and is generally recognized as safe (GRAS) organism [8]. C. glutamicum was listed on the Qualified Presumption of Safety (QPS) list of European Food Safety Authority (EFSA) and the concerns for the safety of C. glutamicum when used for biotechnological process are rarely considered [9]. Recently, this microorganism has shown remarkable potential of biological production of various nutrients including organic acids, vitamins, and amino acids [10][11][12]. Therefore, the importance of C. glutamicum as a role of industrial workhorse in the fields of medicine, biotechnology, and agriculture has been more emphasized.
The safety of C. glutamicum and the three amino acid additives produced through the biotechnological process in C. glutamicum has been evaluated for decades. However, the safety of amino acid additives such as L-threonine, L-tryptophan, and L-valine fermentation products produced by C. glutamicum and other unknown impurities and https://doi.org/10.1016/j.toxrep.2020.01.013 Received 5 November 2019; Received in revised form 23 January 2020; Accepted 26 January 2020

Bacterial reverse mutation test (Ames test)
Under GLP regulation, the Ames test was performed (referencing the OECD Guideline 471 [16], Maron and Ames [17], Vogel and Bonner [18], Green and Muriel [19], and Kilbey et al. [20]) with minor modifications as follows: five histidine auxotroph strains of S. typhimurium TA100, TA1535, TA98, TA1537, TA102, and a tryptophan auxotroph strain of E. coli WP2 uvrA were obtained from Molecular Toxicology Inc. (NC, USA) and were cultured in maintenance or growth media in accordance with the provider's instructions. The mutagenic activity of each test compound was assessed in the absence and presence of external metabolic activation system from rat livers (S9 fraction) using the direct plate incorporation method. For the plating assay, 0.5 mL of S9 mix (or sodium-phosphate buffer, pH 7.4 for non-activation plates), 0.1 mL of bacterial culture (containing approximately 10 8 viable cells), and 0.1 mL of test compound were mixed with 2.0 mL of overlay agar. The contents of each tube were mixed and poured over the surface of a minimal agar plate. The overlay agar was allowed to solidify before incubation. After the top was solidified, plates were inverted and incubated at 37 ± 2°C for 50 ± 2 h and the revertant colonies were counted with unaided eye. The growing concentrations of each test compound were selected based on the results of a range-finding test conducted on the test compound using the five or six test strains in both

In vitro mammalian chromosomal aberration test
The chromosomal aberration test is designed to evaluate the mutagenic potential to induce structural and/or numerical chromosomal aberrations in cultured CHL cells. In vitro chromosomal aberration test was performed according to OECD guidelines 473 [21], Ishidate et al. [22] and Dean and Danford [23] under GLP regulation. Structural abnormalities (g, gap of chromatid or chromosome; ctb, break of chromatid; cte, exchange of chromatid; csb, break of chromosome; cse, exchange of chromosome) and polyploidy as chromosomal aberrations were evaluated in cultured CHL cells in the presence and absence of exogenous metabolic activation system, consisting of the cofactor-supplemented post-mitochondrial fraction (S9) of liver homogenates from rats treated with Aroclor 1254 [24]. CHL/IU cells were cultured for 3 days from an aliquot of 5 × 10 4 cells per flask (culture surface, 25 cm 2 ) [25]. Short-term (6-8 hours) and continuous (24 h) treatments were performed as follows: concentrations of L-threonine fermentation product were 500, 1000, and 2000 μg/mL for 6 + S, 6-S, and 24-S; concentrations of L-valine fermentation product were 250, 500, 800, and 1000 μg/mL for 6 + S, 6-S and 24-S; concentrations of L-tryptophan fermentation product were 0, 87.5, 125, 175, 250, 310, 350, and 500 μg/mL for 6 + S, 6-S and 24-S. Approximately 22 h after treatment, 50 μL of colchicine solution was added to each culture (final concentration of 1 μM) and incubated for 2 h for mitotic arrest. The mitotic cells were detached by gentle shaking. The medium containing mitotic cells were centrifuged, and the cell pellets were resuspended in 75 mM potassium chloride solution for hypotonic treatment. Then cells were fixed with fixative (methanol:glacial acetic acid = 3:1 v/v) three times and slides were prepared by the air-drying method. Slides were stained with 5 % Giemsa solution. Two slides were prepared for each culture. One hundred and fifty metaphases per flask (300 metaphases per concentration) were evaluated for chromosome aberrations. The results were expressed as frequency (%) of metaphases with structural or numerical aberrations per 300 metaphases. Relative increase in cell count (RICC) was used as an indicator of concurrent cytotoxicity to determine the high concentration. With the cell counts, RICC was calculated as follows and used as an indicator of concurrent cytotoxicity. The selection of maximum concentration was based on cytotoxicity, and the highest concentration aimed to achieve 55 ± 5% cytotoxicity. All concentrations of test compounds as well as the concurrent negative (vehicle) and the positive control (benzo[a]pyrene (B[a]P) and 4-nitroquinoline-1-oxide (4NQO) for L-valine and L-tryptophan  fermentation products; Mitomycin C (MMC) and Cyclophosphamide monohydrate (CPA) for L-threonine fermentation product), were evaluated using duplicate cultures per concentration.

Acute oral toxic class method study
The present study was performed to assess the toxicity of L-threonine fermentation product in Wistar rats and L-valine and L-tryptophan fermentation products in Sprague-Dawley rats following a single oral administration acute toxic method according to OECD guideline 420 [26]. Stepwise procedure with two fixed doses (a sighting test in one female at 300 mg/kg b.w. and in one female at 2000 mg/kg b.w., and further main test in four females at 2000 mg/kg b.w.) was performed. In two fixed doses, six females were treated with L-threonine, L-valine, and L-tryptophan fermentation products by oral gavage administration. Before dosing, animals were fasted overnight and then the test compound was directly administered into stomach using a syringe tube with a feeding needle. Food was given in 3 or 4 h after administration. All animals were observed for mortality and clinical signs every hour for 6 h after dosing during the first 24 h and then once daily for a total of 14 days. Body weights were recorded on day 1(prior to the administration), 2, 4, 8, and 15 after dosing of L-valine and L-tryptophan fermentation products, while they were recorded on day 1(prior to the administration), 8 and 15 after dosing of L-threonine fermentation product. At study termination, all animals were euthanized by CO 2 inhalation and their organs were then collected for macroscopic necropsy examination.

Statistical analysis
Statistical analysis was performed using SPSS Statistics version 22. The level of significance was taken as P ≤ 0.05. Fisher's exact test was used to compare the frequencies of aberrant metaphase between the negative control and treated groups for the chromosomal aberration test. Statistical analysis for acute oral toxicity was not performed as number of animals was insufficient for analysis.

Bacterial reverse mutation test (Ames test)
Mutagenicity tests were performed to evaluate the test compound's potential to induce reverse mutation in the five histidine auxotroph strains of S. typhimurium TA100, TA102, TA1535, TA98, TA1537 and a tryptophan auxotroph strain of E. coli WP2 uvrA in the presence and absence of exogenous metabolic activation system.
As shown in Tables 2 and 3, L-threonine and L-valine fermentation products showed no substantial increases in numbers of revertants per plate at concentration levels (12, 37, 111, 333, 1000, and 3000 μg/plate for L-threonine fermentation product; 50, 150, 500, 1500, and 5000 μg/ plate for L-valine and L-tryptophan fermentation products) in TA100, TA1535, TA98, TA1537 and WP2 uvrA in either the presence or absence of metabolic activation of S9 mix. Also, L-tryptophan fermentation product showed no substantial increases in numbers of revertants per plate in TA100, TA102, TA1535, TA98 and TA1537 strains at concentration levels (50, 150, 500, 1500, and 5000 μg/plate) in either the presence or absence of metabolic activation of S9 mix (Table 4). However, in WP2 uvrA both in the presence and absence of metabolic activation system in Table 4, L-tryptophan induced significant increases (P ≤ 0.05) of the revertants per plate in WP2 uvrA. In addition, the mean revertant of the positive control for each test strain exhibited a clear increase over the mean revertant of the negative control for that strain. There was no indication of mutagenicity at all concentrations tested.

In vitro mammalian chromosomal aberration test
As shown in Tables 5-10, cytotoxicity was observed at the highest concentrations of L-valine (24-S) and L-tryptophan (6-S and 24-S). There were no statistical significant increases in the frequencies of aberrant metaphases with structural (< 5.0 % in both with gap (+gap) and without gap (-gap)) or numerical (almost 0 %) aberrations at any concentrations of the test compounds compared to the concurrent negative control. In addition, B[a]P, 4-NQO, MMC or CPA, induced a clear increase in the frequency of aberrant metaphases with structural aberrations as shown in Tables 5-10.

Acute oral toxicity
Under the present laboratory conditions, there were no mortalities, no clinical signs, no changes of body weight, and no macroscopic findings in necropsy at the dose of 300 mg/kg b.w. and 2000 mg/kg b.w. as shown in Tables 11-13. Based on the results of this study, when each test compound was dosed to rats by a fixed dose procedure method, all test compounds were categorized as GHS category 5/unclassified.

Discussion
The present study was performed to evaluate the toxicity of the  To evaluate potential genotoxicity of the three amino acids fermentation products, bacterial reverse mutation tests and chromosomal aberration tests were carried out. In the Ames assay investigating the potential of the three amino acid additives to induce gene mutations at concentrations of up to 3000 μg/plate (L-threonine) and 5000 μg/plate (L-valine and L-tryptophan fermentation products), no biologically-relevant increase in revertant colony numbers was found in any of the tested strains S. typhimurium TA100, TA1535, TA98, TA1537 and in combination with E. coli WP2 uvrA in the presence or absence of metabolic activation system. Also, a mammalian cytogenetic assay in cultured CHL cells for the direct observation in identifying chromosomal aberrations and no statistically-significant or concentration-dependent increase was found in the frequencies of aberrant metaphases with structural or numerical aberrations at all concentrations of the test compound compared to the concurrent negative control. Interestingly, a significant increase of the revertants per plate was found in WP2 uvrA with treatment of L-tryptophan fermentation product. It was considered that the addition of L-tryptophan fermentation product (Table 1) could lead to false positives in WP2 uvrA, resulting in the increases of revertants per plate [27]. It was considered that increases in numbers of colonies in WP2 uvrA may have been due to the content of L-tryptophan (63.09% in total) as shown in Table 1. To double-check the increase of the revertants in WP2 uvrA by L-tryptophan fermentation product, S.  typhimurium TA102, one of recommended strains in the OECD 471 Guideline, was tested at concentration levels (50, 150, 500, 1500, and 5000 μg/plate) of L-tryptophan fermentation product. As shown in Table 4, there was no increase in the number of revertants compared to the negative control in the TA102 strain. However, the positive control as 2-aminoanthracene showed a 6-fold increase of revertants compared to the negative control in the same strain. Therefore, the L-tryptophan fermentation product is non-mutagenic under the conditions of the present study. In conclusion, the three amino acid additives are nonmutagenic under the laboratory conditions of the present study.
In this acute oral toxicity study, two separate independent GLPcertified research institutes, one in the Republic of Korea and the other in the UK. Each independently conducted the same acute oral toxicity tests at the sponsor's request. The animals were sourced from two different suppliers. The results of the acute oral toxicity test demonstrated that the three amino acid additives categorized as GHS category 5/ unclassified. No mortalities, clinical signs, change of body weight, and gross abnormal necropsy findings were observed as results of the three amino acid additives at the dose of 2000 mg/kg in both Sprague-Dawley in Chemon in the Republic of Korea and Wistar rats in Envigo in the UK. These results suggest that the three amino acid additives did not demonstrate toxicity regardless of the animal strains being sourced from two different outbred rodent suppliers. In this study, the three amino acid additives at dose of 2000 mg/kg had no adverse effect on the tested rats through Day 1-14 in clinical observations, changes of body weights, and necropsy findings. Therefore, this study indicates that the three amino acid additives do not cause acute toxicity effects at the doses tested and the LD 50 value was > 2000 mg/kg. The determination of LD 50 from acute toxicity studies is usually the initial step to serve as the basis for classification and provides initial information on the mode of toxic action of a test compound. According to the chemical labeling and classification of acute systemic toxicity recommended by the OECD, the three amino acid additives were assigned as class 5/ unclassified at which dose level the animals are expected to survive under the present laboratory conditions. Furthermore, the data of the acute toxicity study should be further analyzed to increase the confidence in establishing the compounds safety as ingredients or for other biological uses. However, further evaluative toxicity studies should be performed to investigate toxicological profiles such as the target organ or delayed toxicity for the three amino acid additives for up to 90 days in rats, although there were no toxicological effects found in clinical signs, changes of body weights, and necropsy findings under the present laboratory conditions in the acute toxicity study.
In addition, L-threonine, L-valine and L-tryptophan on the list of European Food Safety Authority (EFSA) produced by an aerobic fermentation process using C. glutamicum, are generally recognized as safe (GRAS) for humans and food producing animals and testing showed that there is no exposure risk to humans consuming tissues or products from the target animals [28][29][30]. As shown in Table 1, toxicological changes in gene mutations, chromosome aberration and acute oral toxicity were evaluated in the presence of other unknown impurities and derivatives from C. glutamicum in amino acid additives. Under the  present laboratory conditions, C. glutamicum was found to be non-mutagenic, non-clastogenic in genotoxicity, and non-toxic in the acute oral toxicity test.

Conclusion
The present study and the results presented herein, support the safety of the three amino acid additives with C. glutamicum in terms of genotoxicity and acute oral toxicity in accordance with the OECD guidelines and the principles of GLP. For the first time, these results show that the three amino acid additives with C. glutamicum are safe with no adverse effects and may be applied as an ingredient in products for food-producing animals or other biological uses.

Table 11
Body weights of the three amino acids fermentation product-treated animals. The day of administration was designated as Day 1.
Data are expressed as mean ± S.D.

Declaration of Competing Interest
The authors declare that there is no conflict of interest.

Table 13
Clinical signs of the three amino acids fermentation product treated animals.