Evaluation of the herbicide glyphosate, (aminomethyl)phosphonic acid, and glyphosate-based formulations for genotoxic activity using in vitro assays.

Glyphosate, the most heavily used herbicide world-wide, is applied to plants in complex formulations that promote absorption. The National Toxicology Program reported in 1992 that glyphosate, administered to rats and mice at doses up to 50,000 ppm in feed for 13 weeks, showed little evidence of toxicity, and no induction of micronuclei was observed in the mice in this study. Subsequently, mechanistic studies of glyphosate and glyphosate-based formulations (GBFs) that have focused on DNA damage and oxidative stress suggest that glyphosate may have genotoxic potential. However, few of these studies directly compared glyphosate to GBFs, or effects among GBFs. To address these data gaps, we tested glyphosate, glyphosate isopropylamine (IPA), and (aminomethyl)phosphonic acid (AMPA, a microbial metabolite of glyphosate), 9 high-use agricultural GBFs, 4 residential-use GBFs, and additional herbicides (metolachlor, mesotrione, and diquat dibromide) present in some of the GBFs in bacterial mutagenicity tests, and in human TK6 cells using a micronucleus assay and a multiplexed DNA damage assay. Our results showed no genotoxicity or notable cytotoxicity for glyphosate or AMPA at concentrations up to 10 mM, while all GBFs and herbicides other than glyphosate were cytotoxic, and some showed genotoxic activity. An in vitro to in vivo extrapolation of results for glyphosate suggests that it is of low toxicological concern for humans. In conclusion, these results demonstrate a lack of genotoxicity for glyphosate, consistent with observations in the NTP in vivo study, and suggest that toxicity associated with GBFs may be related to other components of these formulations.

Using the HepaCometChip Assay for Broad-Spectrum DNA Damage Analysis.

Exposure to DNA damaging agents can lead to mutations that cause cancer. The liver is particularly vulnerable because it contains high levels of Cytochrome P450 enzymes that can convert xenobiotics into DNA reactive metabolites that form potentially carcinogenic bulky DNA adducts. As such, current requirements for preclinical testing include in vivo testing for DNA damage in the liver, which often requires many animals. Given that efforts are underway in many countries to reduce or eliminate the use of animals in research, there is a critical need for fast and robust in vitro tests to discern whether xenobiotics or potential pharmaceutical agents can damage the hepatocyte genome. One possible approach is to leverage the alkaline comet assay, which is used to assess genotoxicity based on the ability of damaged DNA to become free to migrate toward the anode during electrophoresis. The comet assay, however, has several limitations. The assay is (i) slow and (ii) vulnerable to experimental noise, (iii) it is difficult to detect bulky DNA adducts since they do not directly affect DNA migration, and (iv) cell types typically used do not have robust metabolic capacity. To address some of these concerns, we have developed the "HepaCometChip" (a.k.a. the HepaRG CometChip), wherein metabolically competent cells are incorporated into a higher throughput CometChip platform. Repair trapping is used to increase sensitivity for bulky lesions: undetectable bulky lesions are converted into repair intermediates (specifically, single-strand breaks) that can be detected with the assay. Here, we describe a protocol for performing the HepaCometChip assay that includes handling and dosing of HepaRG cells and performing the CometChip assay. With its higher throughput, ability to capture metabolic activation, and sensitivity to bulky lesions, the HepaCometChip offers a potential alternative to the use of animals for genotoxicity testing. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: HepaRG cell culturing and dosing Basic Protocol 2: CometChip assay.

Bacterial mutagenicity assays: Vehicle and positive control results from the standard Ames assay, the 6- and 24-well miniaturized plate incorporation assays and the Ames II™ assay.

Bacterial mutation assays are conducted routinely as part of the safety assessment of new chemicals. The OECD Test Guideline (TG) 471 describes the conduct of the standard agar plate Ames assay, required for regulatory submissions. Higher throughput non-OECD 471 TG assays, such as the miniaturized plate incorporation and Ames II™ assays, can be used for prescreening purposes. We have compiled historical vehicle and positive control data generated using these methods. The historical database is comprised from experiments spanning 9 years and includes >1000 experiments from the standard Ames assay using the plate incorporation and pre-incubation methods (TA98, TA100, TA1535, TA1537, and WP2 uvrA), >50 experiments from the 6-well (TA98, TA100, TA1535, TA97a, and WP2 uvrA) and >100 experiments from the 24-well (TA98, TA100, TA102, TA1535, TA1537, and TA97a) plate incorporation assays, and >1000 experiments from the Ames II™ assay (TA98 and TAMix). Although miniaturization to a 24-well format made the measurement of control revertant colonies in TA1537 and TA1535 more difficult; this can be overcome by using an alternative strain with a higher spontaneous reversion rate (i.e., using TA97a instead of TA1537) or by increasing the number of replicate wells to 12 (for TA1535). All three miniaturized methods, including the Ames II™ assay, were responsive to known mutagens and the responses were reproducible over years of use. These data demonstrate the excellent reproducibility of the standard and miniaturized bacterial mutation assays using positive control chemicals. Environ. Mol. Mutagen. 57:483-496, 2016. © 2016 Wiley Periodicals, Inc.

Combination comet/micronucleus assay validation performed by BioReliance under the JaCVAM initiative.

In the international validation study of the in vivo rat alkaline comet assay (comet assay), the Japanese Center for the Validation of Alternative Methods (JaCVAM) provided three coded chemicals to BioReliance, 1,3-dichloropropene, ethionamide and busulfan, to be tested in a combined in vivo comet/micronucleus assay. Induction of DNA damage (comet) in liver, stomach and jejunum (1,3-dichloropropene only) cells, and induction of MNPCEs in bone marrow, were examined in male Sprague-Dawley (Hsd:SD) rats following oral administration of the test chemical for three consecutive days. A dose range finding (DRF) test was performed with each chemical to determine the maximum tolerated dose (MTD). Based on the results of the DRF test; 1,3-dichloropropene was tested at 50, 100 and 200 mg/kg/day; ethionamide was tested at 125, 250 and 500 mg/kg/day, and busulfan was tested at 10, 20 and 40 mg/kg/day. The results indicated that 1,3-dichloropropene induced DNA damage only in liver cells at all three test article doses, while no effects were observed in the stomach and jejunum cells. Additionally, it did not increase MNPCEs in the bone marrow. 1,3-Dichloropropene was concluded to be negative in the MN assay but positive in the comet assay. Ethionamide did not induce DNA damage in liver. However, in stomach, statistically significant decreases (although still within historical range) in % tail DNA at all test article doses compared to the vehicle control were observed. There was no increase in MNPCEs in the bone marrow. Thus, ethionamide was concluded to be negative in the comet/MN combined assay. Busulfan did not induce DNA damage in any of the organs tested (liver and stomach) but it did induce a significant increase in MNPCEs in the bone marrow. Busulfan was concluded to be negative in the comet assay but positive in the MN assay.

Prevalidation study of the Syrian hamster embryo (SHE) cell transformation assay at pH 7.0 for assessment of carcinogenic potential of chemicals.

The European Centre for the Validation of Alternative Methods (ECVAM) has organised an interlaboratory prevalidation study on the Syrian hamster embryo (SHE) cell transformation assay (CTA) at pH 7.0 for the detection of rodent carcinogens. The SHE CTA at pH 7.0 has been evaluated for its within-laboratory reproducibility, transferability and between-laboratory reproducibility. Four laboratories using the same basic protocol with minor modifications participated in this study and tested a series of six coded-chemicals: four rodent carcinogens (benzo(a)pyrene, 3-methylcholanthrene, 2,4-diaminotoluene and o-toluidine HCl) and two non-carcinogens (anthracene and phthalic anhydride). All the laboratories found the expected results with coded chemicals except for phthalic anhydride which resulted in a different call in only one laboratory. Based on the outcome of this study, it can be concluded that a standardised protocol is available that should be the basis for future use. This protocol and the assay system itself are transferable between laboratories and the SHE CTA at pH 7.0 is reproducible within- and between-laboratories.

Photo catalogue for the classification of cell colonies in the Syrian hamster embryo (SHE) cell transformation assay at pH 6.7.

This catalogue is a display of Syrian hamster embryo (SHE) cell colony photos representative of the cell transformation assay (CTA) carried out at pH 6.7. It is intended as a visual aid for the identification and the scoring of cell colonies in the conduct of the assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the assay results.

Prevalidation study of the Syrian hamster embryo (SHE) cell transformation assay at pH 6.7 for assessment of carcinogenic potential of chemicals.

The Syrian hamster embryo (SHE) cell transformation assay (CTA) is an important in vitro method that is highly predictive of rodent carcinogenicity. It is a key method for reducing animal usage for carcinogenicity prediction. The SHE assay has been used for many years primarily to investigate and identify potential rodent carcinogens thereby reducing the number of 2-year bioassays performed in rodents. As for other assays with a long history of use, the SHE CTA has not undergone formal validation. To address this, the European Centre for the Validation of Alternative Methods (ECVAM) coordinated a prevalidation study. The aim of this study was to evaluate the within-laboratory reproducibility, test method transferability, and between-laboratory reproducibility and to develop a standardised state-of-the-art protocol for the SHE CTA at pH 6.7. Formal ECVAM principles for criteria on reproducibility (including the within-laboratory reproducibility, the transferability and the between-laboratories reproducibility) were applied. In addition to the assessment of reproducibility, this study helped define a standard protocol for use in developing an Organisation for Economic Co-operation and Development (OECD) test guideline for the SHE CTA. Six compounds were evaluated in this study: benzo(a)pyrene, 3-methylcholanthrene, o-toluidine HCl, 2,4-diaminotoluene, phthalic anhydride and anthracene. Results of this study demonstrate that a protocol is available that is transferable between laboratories, and that the SHE CTA at pH 6.7 is reproducible within- and between-laboratories.

Recommended protocol for the Syrian hamster embryo (SHE) cell transformation assay.

The Syrian hamster embryo (SHE) cell transformation assay (CTA) is a short-term in vitro assay recommended as an alternative method for testing the carcinogenic potential of chemicals. SHE cells are "normal" cells since they are diploid, genetically stable, non-tumourigenic, and have metabolic capabilities for the activation of some classes of carcinogens. The CTA, first developed in the 1960s by Berwald and Sachs (1963,1964) [3,4], is based on the change of the phenotypic feature of cell colonies expressing the first steps of the conversion of normal to neoplastic-like cells with oncogenic properties. Pienta et al. (1977) [22] developed a protocol using cryopreserved cells to enhance practicality of the assay and limit sources of variability. Several variants of the assay are currently in use, which mainly differ by the pH at which the assay is performed. We present here the common version of the SHE pH 6.7 CTA and SHE pH 7.0 CTA protocols used in the ECVAM (European Centre for the Validation of Alternative Methods) prevalidation study on CTA reported in this issue. It is recommended that this protocol, in combination with the photo catalogues presented in this issue, should be used in the future and serve as a basis for the development of the OECD test guideline.

Assessment of genotoxicity induced by 7,12-dimethylbenz(a)anthracene or diethylnitrosamine in the Pig-a, micronucleus and Comet assays integrated into 28-day repeat dose studies.

As part of the Stage 3 of the Pig-a international trial, we evaluated 7,12-dimethylbenz(a)anthracene (DMBA) for induction of Pig-a gene mutation using a 28-day repeat dose study design in Sprague-Dawley rats. In the same study, chromosomal damage in peripheral blood and primary DNA damage in liver were also investigated by the micronucleus (MN) assay and the Comet assay, respectively. In agreement with previously published data (Dertinger et al., [2010]: Toxicol Sci 115:401-411), DMBA induced dose-dependent increases of CD59-negative erythrocytes/reticulocytes and micronucleated reticulocytes (MN-RETs). However, there was no significant increase in DNA damage in the liver cells when tested up to 10 mg/kg/day, which appears to be below the maximum tolerated dose. When tested up to 200 mg/kg/day in a follow-up 3 dose study, DMBA was positive in the liver Comet assay. Additionally, we evaluated diethylnitrosamine (DEN), a known mutagen/hepatocarcinogen, for induction of Pig-a mutation, MN and DNA damage in a 28-day study. DEN produced negative results in both the Pig-a mutation assay and the MN assay, but induced dose-dependent increases of DNA damage in the liver and blood Comet assay. In summary, our results demonstrated that the Pig-a mutation assay can be effectively integrated into repeat dose studies and the data are highly reproducible between different laboratories. Also, integration of multiple genotoxicity endpoints into the same study not only provides a comprehensive evaluation of the genotoxic potential of test chemicals, but also reduces the number of animals needed for testing, especially when more than one in vivo genotoxicity tests are required.

An international validation study of a Bhas 42 cell transformation assay for the prediction of chemical carcinogenicity.

The Bhas 42 cell transformation assay is a sensitive short-term system for predicting chemical carcinogenicity. Bhas 42 cells were established from BALB/c 3T3 cells by the transfection of v-Ha-ras gene and postulated to have acquired an initiated state in the two-stage carcinogenesis theory. The Bhas 42 cell transformation assay is capable of detecting both tumor-initiating and tumor-promoting activities of chemical carcinogens. The full assay protocol consists of two components, the initiation assay and the promotion assay, to detect the initiating activity and the promoting activity, respectively. An international study was carried out to validate this cell transformation assay in which six laboratories from three countries participated. Twelve coded chemicals were examined in total and each chemical was tested by three laboratories. In the initiation assay, concordant results were obtained by three laboratories for eight out of ten chemicals and in the promotion assay, concordant results were achieved for ten of twelve chemicals. The positive results were obtained in all three laboratories with the following chemicals: 2-acetylaminofluorene was positive in both initiation and promotion assays; dibenz[a,h]anthracene was positive in the initiation assay; sodium arsenite, lithocholic acid, cadmium chloride, mezerein and methapyrilene hydrochloride were positive in the promotion assay. o-Toluidin hydrochloride was positive in the both assays in two of the three laboratories. d-Mannitol, caffeine and l-ascorbic acid were negative in both assays in all the laboratories, and anthracene was negative in both assays in two of the three laboratories except one laboratory obtaining positive result in the promotion assay. Consequently, the Bhas 42 cell transformation assay correctly discriminated all six carcinogens and two tumor promoters from four non-carcinogens. Thus, the present study demonstrated that the Bhas 42 cell transformation assay is transferable and reproducible between laboratories and applicable to the prediction of chemical carcinogenicity. In addition, by comparison of the present results with intra-laboratory data previously published, within-laboratory reproducibility using the Bhas 42 cell transformation assay was also confirmed.

Syrian hamster embryo (SHE) cell transformation assay with conditioned media (without X-ray irradiated feeder layer) using 2,4-diaminotoluene, 2,6-diaminotoluene and chloral hydrate.

The Syrian hamster embryo (SHE) cell transformation assay has traditionally been conducted with a feeder layer of X-ray irradiated cells to provide growth support to the target cells seeded in low numbers. The feeder layer of cells consists of X-ray irradiated cells which are still viable but unable to replicate. We have tried seeding the target cells in conditioned media prepared from the stock culture flasks in lieu of plating them on a feeder layer. Three SHE cell isolates were tested to investigate the feasibility of this approach. With freshly prepared conditioned medium (LeBoeuf's Dulbecco's Modified Eagle's Medium with 2 mM L-glutamine and 20% fetal bovine serum), used within 2 weeks of preparation, there was essentially no difference in the number of target cell colonies in the conditioned medium and in the plates with the X-ray irradiated feeder cell layer. The plating efficiencies of the vehicle controls were within the historical range for the standard SHE cell transformation assay. In each experiment, the positive control benzo(a)pyrene [B(a)P] elicited a significant increase in morphological transformation frequency (MTF), with or without feeder cells. Three compounds, 2,4-diaminotoluene (2,4-DAT), 2,6-diaminotoluene (2,6-DAT), and chloral hydrate were tested in the SHE cell transformation assay without an X-ray irradiated feeder layer and using a 7-day exposure regimen. The results were comparable to those reported in the published literature using the standard methodology with feeder cells, with 2,4-DAT and chloral hydrate eliciting a significant increase in MTF, and 2,6-DAT not eliciting any increase in MTF. The results of this study demonstrate the feasibility of conducting the SHE cell transformation assay without the use of an X-ray irradiated feeder layer, thereby simplifying the test procedure and facilitating the scoring of morphologically transformed colonies.