Optimization of the Ames RAMOS test allows for a reproducible high-throughput mutagenicity test

Science of the Total Environment 717 (2020) 137168

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Optimization of the Ames RAMOS test allows for a reproducible highthroughput mutagenicity test Kira Kauffmann a, Felix Werner a, Alexander Deitert a, Julian Finklenburg a, Julia Brendt b, Andreas Schiwy b,1, Henner Hollert b,1, Jochen Büchs a,⁎ a b

AVT-Chair for Biochemical Engineering, RWTH Aachen University, Forckenbeckstraße 51, 52074 Aachen, Germany Institute for Environmental Research, Department of Ecosystem Analysis, Worringerweg 1, 52074 Aachen, Germany

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Newly developed Ames RAMOS test allows online detection of mutagenicity. • Observed variability due to histidine transfer from preculture could be eliminated. • A higher initial histidine concentration increases sensitivity of Ames RAMOS test. • A lower cultivation temperature increases sensitivity of Ames RAMOS test. • Successful generation of dose response data in high throughput Ames RAMOS test

a r t i c l e

i n f o

Article history: Received 20 December 2019 Received in revised form 5 February 2020 Accepted 5 February 2020 Available online 07 February 2020 Editor: Damia Barcelo Keywords: Ames test RAMOS Oxygen transfer rate Salmonella typhimurium Histidine Mutagenicity

a b s t r a c t The Ames test is one of the most widely used mutagenicity tests. It employs histidine auxotrophic bacteria, which can mutate back to histidine prototrophy and, thus, grow on a histidine deficient medium. These mutants develop predominantly after adding a mutagenic compound during an initial growth phase on 1 mg/L histidine. In the established test systems, an endpoint determination is performed to determine the relative number of mutants. An alternative Ames test, the Ames RAMOS test, has been developed, which enables the online detection of mutagenicity by monitoring respiration activity. The reproducibility of the newly developed test system was investigated. A strong dependence of the test results on the inoculum volume transferred from the preculture was found. The more inoculum was needed to reach the required initial OD, the more mutagenic a positive control was evaluated. This effect was attributed to the histidine transfer from the preculture to the original Ames RAMOS test. The same problem is evident in the Ames fluctuation test. High reproducibility of the Ames RAMOS test could be achieved by performing the preculture on minimal medium with a defined histidine concentration and termination after histidine depletion. By using 5 mg/L initial histidine within the minimal medium, a higher separation efficiency between negative control and mutagenic samples could be achieved. This separation efficiency could be further increased by lowering the cultivation temperature from 37 to 30 °C, i.e. lowering the maximum growth rate. The optimized Ames RAMOS test was then transferred into a 48-well microtiter plate format (μRAMOS) for obtaining a high throughput test. The online detection of mutagenicity leads to a

Abbreviations: DMSO, dimethyl sulfoxide; FAU, formazine attenuation units; OD, optical density; OTR, oxygen transfer rate; RAMOS, respiration activity monitoring system. ⁎ Corresponding author. E-mail addresses: [email protected] (K. Kauffmann), [email protected] (F. Werner), [email protected] (A. Deitert), julian.fi[email protected] (J. Finklenburg), [email protected] (J. Brendt), [email protected] (A. Schiwy), [email protected] (H. Hollert), [email protected] (J. Büchs). 1 Current address: Department of Evolutionary Ecology and Environmental Toxicology, Goethe University Frankfurt, Max-von-Laue-Str. 13, 60438 Frankfurt am Main, Germany.

https://doi.org/10.1016/j.scitotenv.2020.137168 0048-9697/© 2020 Elsevier B.V. All rights reserved.

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K. Kauffmann et al. / Science of the Total Environment 717 (2020) 137168

reduction of working time in the laboratory. Due to the optimization of reproducibility and the increase in separation efficiency, a sound mutagenicity evaluation, even of weak mutagenic compounds, can be achieved. © 2020 Elsevier B.V. All rights reserved.

1. Introduction To avoid the exposure of humans and the environment with mutagens, newly developed chemicals should be tested for being nonmutagenic (Ames, 1979; Ji et al., 2017). The Ames test is a worldwide known and widely used mutagenicity test for the evaluation of potential mutagens, both in the environment and industry (Mortelmans, 2019; Mortelmans and Zeiger, 2000; Zeiger, 2019). An in vitro test in bacteria as the Ames test is required for the assessment of chemicals by the European Chemicals Regulation (REACH) as described in Regulation (ec) no 1907/2006 of The European Parliament and of the Council (2006) and the Japanese Chemical Substances Control Law (CSCL) (Ji et al., 2017). Additionally, the test was very frequently used in the last decades for the evaluation of mutagenicity in both, surface water and drinking water samples (Di Paolo et al., 2016; Reifferscheid et al., 2012) Recently, a consensus battery of effect-based methods (including the Ames test) was suggested by the EU project Solutions. The NORMAN (Network of reference laboratories, research centres and related organisations for monitoring of emerging environmental substances) network and the CIS (Common Implementation Strategy) working group of the European Commission for effect based methods are working on an implementation into the Water Framework Directive (WFD) (Brack et al., 2019, 2017). Various types of Ames tests exist. The original Ames test on agar plates was developed by Bruce Ames (Ames et al., 1973a) and is still widely used (Zeiger, 2019). It is standardized by OECD Guideline 471 (1997). Based on this original test system, the Ames fluctuation test in microtiter plates was developed (Reifferscheid et al., 2012) and standardized by ISO Guideline 11350 (2012). Both test systems are based on the same auxotrophic bacterial strains of Salmonella typhimurium or Escherichia coli. In this study, histidine auxotrophic Salmonella typhimurium strains, TA 100 and TA 98, are used. Each contains specific single base mutations in the histidine operon (McCann et al., 1975; Mortelmans and Zeiger, 2000). Due to spontaneous as well as induced mutations, the cells can regain histidine prototrophy and can grow on histidine deficient medium. These cells are called revertants. The more mutagenic potential a compound has, the more revertans occur (Ames et al., 1973a; Maron and Ames, 1983). The mutations are manifested during replication, thus, during the growth of the bacteria. Hence, 1 mg/L initial histidine is added to the used minimal medium to enable a few cell divisions and to allow the mutations to manifest during growth (ISO 11350, 2012; OECD 471, 1997). As described by Kauffmann et al. (2019), an alternative new type of Ames test, the Ames RAMOS test, has recently been developed. It allows mutagenicity detection by online monitoring of the respiration activity of S. typhimurium using a respiration activity monitoring system (RAMOS) in shake flasks (Anderlei and Büchs, 2001; Anderlei et al., 2004). Due to spontaneous mutations back to histidine prototrophy, the respiration activity, measured by the oxygen transfer rate (OTR), starts to rise at some point in the histidine deficient medium. When a mutagenic compound is added, an earlier increase in respiration activity is visible (Kauffmann et al., 2019). All other published Ames test variants, including the standardized Ames test on agar plates and the Ames fluctuation test, are endpoint determinations providing no information about actual growth and metabolic activity of the bacteria. In the new Ames RAMOS test, no additional endpoint determination is necessary. At the same time, online monitoring of respiration activity allows for a deeper investigation of the mechanisms taking place during the Ames test and an online determination of cytotoxicity.

Both established test systems, the Ames test on agar plates as well as the Ames fluctuation test have been intensively validated and allow the evaluation of possible mutagens. Nevertheless, a certain variability is well known (Aeschbacher et al., 1983; Agnese et al., 1984; Cheli et al., 1980; Knuiman et al., 1987; Levy et al., 2019; Margolin et al., 1984; Maron and Ames, 1983; Reifferscheid et al., 2012; Seiler, 1983). Thus, also the Ames RAMOS test was investigated in terms of reproducibility. Reasons for an observed variability were investigated to obtain an improved test protocol for reproducible test results. The preculture of the Ames test is conducted on a complex medium. Variabilities of complex media have been published and discussed by Diederichs et al. (2014). According to OECD Guideline 471 (1997), the inoculum volume transferred from the complex preculture to the Ames test on agar plates is kept constant. In contrast, according to the guideline for the Ames fluctuation test (ISO 11350, 2012) the initial optical density (OD) in the test has to be kept constant. This leads to a varying amount of complex preculture added to the test (exposure) culture, due to varying final ODs in the preculture. This second inoculation strategy was initially also used for the original Ames RAMOS test and, thus, was investigated to develop a reproducible test system. Since histidine is present in the complex preculture medium, histidine might also be transferred to the Ames fluctuation test and the Ames RAMOS test proportional to the inoculum volume. This leads to an additional amount of histidine in surplus to the 1 mg/L histidine initially added to the test medium, especially as the ISO Guideline 11350 (2012) does not require a washing step to remove any excess histidine. In this study, both the shake flask RAMOS as well as a newly developed μRAMOS device in 48-well microtiter plates (Flitsch et al., 2016) were used to investigate the effect of inoculum volume on the results of the original Ames RAMOS test. S. typhimurium TA 100 was used as model organism and was tested with a negative control and a positive control (nitrofurantoin). The goal was to improve the test system in terms of standardization, reproducibility and sensitivity. To increase the sensitivity, also the specifically added histidine concentration in the test culture as well as the cultivation temperature and, thus, the maximum growth rate, were investigated. For the first time, a special focus is put on the histidine amount in the preculture medium and its transfer into the test culture. Also the influence of the cultivation temperature on the test results has not been investigated before. So far, the newly developed test system had only been tested with one negative and one positive control without the addition of a metabolic activation system like S9 produced from rat liver (Ames et al., 1973b). Likewise, the test procedure in shake flasks is not practicable for recording dose-response curves in a high-throughput mutagenicity test. Hence, the test was downscaled to the μRAMOS system. Using this device, exemplary dose-response curves for four known mutagenic reference compounds for both S. typhimurium TA 100 and TA 98 with and without S9 were obtained. 2. Material & methods During this study, several experiments were conducted to investigate and optimize the Ames RAMOS test. All experiments performed are summarized in Table 1, including the used cultivation systems and media. The different applied preculture strategies are shown in Fig. 1. Precultures on complex medium were terminated in the late exponential phase. They were either directly diluted to a biomass concentration corresponding to 45 FAU with minimal medium (Fig. 1A) or were

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Table 1 Overview about the conducted experiments for investigating and optimizing the Ames RAMOS test. Experiment

T Cultivation (°C) system

Original Ames RAMOS test: Assessment of variability

37

Influence of initial histidine concentration in the test culture

37

Generation of dose response curves with the optimized Ames RAMOS test 30

Shake flasks

Preculture Test culture Shake flasks Preculture Test culture Shake flasks Preculture Microtiter plates Test culture

diluted with the supernatant of another preculture on complex medium prior to the dilution to 45 FAU with minimal medium to mimic a preculture with a lower final OD (Fig. 1B). Precultures on optimized minimal medium were directly terminated after histidine depletion and diluted to 45 FAU with optimized minimal medium (Fig. 1C). All optimization steps were conducted with strain TA 100. Doseresponse curves were generated with strains TA 100 and TA 98. Detailed descriptions of the used strains, media and the cultivation conditions can be found below.

One lot each of the commercially available Ames test strains S. typhimurium TA 100 and TA 98, purchased from Trinova Biochem GmbH (Giessen, Germany) was used.

2.1.1. Cultivation conditions for shake flasks All precultures and the test cultures of the original Ames RAMOS test were conducted in 250 mL shake flasks with a filling volume of 20 mL. They were shaken with 250 rpm at a shaking diameter of 50 mm.

2.1.2. Cultivation conditions for microtiter plates The test culture of the optimized Ames RAMOS test took place in 48well microtiter plates with a filling volume of 2.4 mL/well. They were shaken with 700 rpm at a shaking diameter of 3 mm.

20 µL

Complex medium

Complex medium Minimal medium + 1 mg/L histidine Optimized minimal medium + 20 mg/L histidine Optimized minimal medium + 0, 1, 2.5 or 5 mg/L histidine Optimized minimal medium + 20 mg/L histidine Optimized minimal medium + 5 mg/L histidine

2.1.3. Online monitoring of respiratory activity via oxygen transfer rate (OTR) For all cultivations, the OTR was monitored online. The in-house developed RAMOS system was used to online monitor the OTR in shake flasks (Anderlei and Büchs, 2001). A similar RAMOS device is commercially available from Kühner AG (Birsfelden, Switzerland) or HiTec Zang GmbH (Herzogenrath, Germany). The μRAMOS systems, in-house developed and described by Flitsch et al. (2016), was used to monitor the OTR in 48-well microtiter plates. 2.2. Media compositions and test compounds

2.1. Microorganism and cultivation conditions

A

Medium conditions

All chemicals were obtained from Sigma Aldrich (Darmstadt, Germany) in a high purity of at least 97.5%, if not stated otherwise. 2.2.1. Complex medium for the preculture of the original Ames RAMOS test For the original preculture, a complex medium was used as described in ISO Guideline 11350 (2012). It contained 18.8 g/L Oxoid nutrient broth No. 2 (lot: 2137737, Thermo Fisher Scientific, Waltham, USA), 1.24 g/L NaCl and 50 mg/L ampicillin. All chemicals were dissolved in deionized water. The pH value was adjusted to 7.5 using NaOH. 2.2.2. Minimal medium for the test culture of the original Ames RAMOS test For the original Ames RAMOS test, a minimal medium as described by Kauffmann et al. (2019) was used. It is based on the minimal medium used in the Ames fluctuation test according to ISO Guideline 11350

Termination in late exponential phase

Minimal medium (1 mg/L histidine)

45 FAU

B

20 µL

Complex medium

Dilution with supernatant

OTR

Termination in late exponential phase

Minimal medium (1 mg/L histidine)

Termination in late exponential phase

45 FAU

200 µL

C Optimized minimal medium (20 mg/L histidine)

Termination after histidine depletion

Positive control

Negative control

Time

Exposition phase of the Ames RAMOS test

Optimized minimal medium (0, 1, 2.5, 5 mg/L histidine)

45 FAU

Fig. 1. Overview about applied preculture strategies. Precultures for the Ames RAMOS test according to Kauffmann et al. (2019) were either conducted on complex medium containing 18.8 g/L nutrient broth powder (Oxoid nutrient broth No. 2, pH0 = 7.5, based on ISO Guideline 11350 (2012)) or optimized minimal medium (pH0 = 7.0). Cultivation conditions: S. typhimurium TA 100, 37 °C, 250 rpm, 50 mm shaking diameter, filling volume 20 mL (250 mL flasks).

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(2012) and Reifferscheid et al. (2012). It contained 4.3 g/L D-glucose, 2.2 g/L citric acid-monohydrate, 10.8 g/L K2HPO4, 3.8 g/L NaNH4HPO4·4 H2O, 0.2 g/L MgSO4·7 H2O, 2.6 mg/L D-biotin and 1 mg/L L-histidine. The pH value was adjusted to a value of 7.0 using NaOH. 2.2.3. Optimized minimal medium for the preculture and test culture of the optimized Ames RAMOS test For the optimized Ames RAMOS test, an optimized minimal medium was developed for the preculture and test culture. The optimized minimal medium contained 4.3 g/L D-glucose, 2.2 g/L citric acidmonohydrate, 10.8 g/L K2HPO4, 3.8 g/L NaNH4HPO4·4 H2O, 2.8 g/L MgSO4·7 H2O, 2.6 mg/L D-biotin and a trace element solution. The trace elements solution is described by Wilms et al. (2001) and consisted of 0.11 mg/L ZnSO4·7 H2O, 0.10 mg/L CuSO4·5 H2O, 0.06 mg/L MnSO4·H2O, 0.11 mg/L CoSO4·7 H2O, 8.36 mg/L FeCl3·6 H2O, 0.40 mg/L CaCl2·2 H2O and 6.69 mg/L Na2EDTA·2 H2O. The pH value was adjusted to a value of 7.0 using NaOH. For the preculture, 20 mg/L L-histidine were added. For the test culture 5 mg/L Lhistidine were added. 2.2.4. Test compounds Stocks of test compounds were added to the test culture of the Ames RAMOS test in a ratio of 1:50. As a negative control compound, pure DMSO was used. The positive control compound for S. typhimurium TA 100 without the addition of S9 for optimization of the original Ames RAMOS test was nitrofurantoin. The final test concentration was 0.25 mg/L (1.05 μmol/L). To obtain dose-response curves for reference compounds in the optimized Ames RAMOS test, six different concentrations by diluting a stock solution 1:2 with DMSO to obtain the next lowest concentration, were applied. In the experimental setup without metabolic activation, the following reference compounds were used. Nitrofurantoin with a highest test concentration of 0.5 mg/L (2.1 μmol/L) was used for strain S. typhimurium TA 100. For strain S. typhimurium TA 98, 2nitrofluorene with a highest test concentration of 0.08 mg/L (3.73 μmol/L) was used. To assess the metabolic activation via rat liver S9 for both strains, 2aminoanthracene with a highest test concentration of 0.8 mg/L (4.14 μmol/L) was used. The S9 was obtained as an induced variant. The induction was conducted with beta-naphtoflavone and phenobarbital. The S9-mix was prepared according to Reifferscheid et al. (2012) (30% (v/v) of S9) and added to the test culture in a ratio of 1:30. 2.3. Original Ames RAMOS test For conducting the original Ames RAMOS test (Kauffmann et al., 2019), the preculture was carried out in accordance with ISO Guideline 11350 (2012) and Reifferscheid et al. (2012) as shown in Fig. 1A. 20 mL of complex growth medium were inoculated with 20 μL of cryo stock of TA 100 and cultivated for 7–8 h until the end of the exponential growth phase, determined by the RAMOS device via an online monitoring of the OTR. The OD of the preculture was measured and then diluted to 45 FAU (TA 100) with minimal medium containing 1 mg/L histidine according to Reifferscheid et al. (2012). Subsequently, this bacterial suspension has been applied in the original Ames RAMOS test in shake flasks. It was carried out according to Kauffmann et al. (2019). The controls and samples were added in a ratio of 1:50 to the test culture, resulting in the test concentrations stated in Section 2.2.4. 2.3.1. Assessment of variability To investigate the variability of the original Ames RAMOS test, results of negative and positive controls of strain TA 100, recorded over a period of 10 months, were compared. During the 10 month test period, the original Ames RAMOS test was conducted 8 times only for a negative control, and 8 times for both a negative and positive control,

respectively. For each test, a new preculture was conducted (biological replicates). A slightly varying final OD of the preculture and a defined initial OD result in different inoculum volume of preculture transferred into the test culture. To further investigate the effect of different inoculum volume on the variability of the original Ames RAMOS test, two precultures on complex medium were cultivated in parallel as shown in Fig. 1B. After ending both precultures, one preculture was centrifuged and the resulting supernatant was used to further dilute the second preculture in ratios of 1:1 to 1:2.5. The OD of the resulting cultivation broth was measured and diluted to 45 FAU with minimal medium to conduct the test culture of the original Ames RAMOS test. 2.4. Influence of initial histidine concentration in the test culture For systematically investigating the influence of the initial histidine concentration in the test culture on the Ames RAMOS test results, the optimized preculture strategy as shown in Fig. 1C was conducted. The OD of the preculture was measured and then diluted to 45 FAU with minimal medium containing 0, 1, 2.5 or 5 mg/L histidine before conducting the test culture of the Ames RAMOS test in optimized minimal medium in triplicates. 2.5. Generation of dose response curves with the optimized Ames RAMOS test For the optimized Ames RAMOS test in the μRAMOS device, the optimized preculture strategy as shown in Fig. 1C was applied. The OD of the preculture was measured and then diluted to 45 FAU (TA 100) or 180 FAU (TA 98) with optimized minimal medium containing 5 mg/L histidine before conducting the test culture of the optimized Ames RAMOS test. The test culture of the optimized Ames RAMOS test was carried out as explained in 2.3 using 1.0× concentrated medium for strain TA 100 and 1.2× concentrated medium for strain TA 98. The controls and reference compounds were added in a ratio of 1:50 to the test culture, resulting in the test concentrations stated in Section 2.2.4. Each concentration was measured in biological triplicates, if not stated otherwise. 3. Results and discussion 3.1. Original Ames RAMOS test: Assessment of variability In a previous study (Kauffmann et al., 2019), the principle of a novel Ames test, the Ames RAMOS test, was presented and a proof of principle was performed. By monitoring the respiration activity of S. typhimurium in a RAMOS system (Anderlei and Büchs, 2001; Anderlei et al., 2004), mutagenicity can be determined. The principle of this original Ames RAMOS test is shown in more detail in Fig. 2A for the test culture of strain TA 100 with a negative and positive control. The oxygen transfer rate (OTR) describes the respiration activity correlating with the growth of the bacteria. As can be seen for both, negative and positive control, the OTR rises at the beginning. This is due to the initial growth on histidine. After histidine is depleted, the auxotrophic cells cannot grow anymore, but they still respire (Kauffmann et al., 2019). This leads to a slow decrease in the OTR because of lysing cells. After 18 and 24 h, the OTR of the positive and negative control, respectively, starts to rise again. This is due to the exponential growth of back-mutated cells, which arise during the initial growth phase on the initially added histidine. The more mutants arise, the earlier their exponential growth is visible in the OTR. Thus, the time until the OTR rises (a threshold of 4 mmol/L/h was chosen for comparing the results as described by Kauffmann et al. (2019)) is proportional to the number of mutants, which have reverted to histidine prototrophy. Consequently, the positive control, where cells mutate spontaneously and induced, rises earlier than the negative control, where cells only mutate spontaneously. The difference between the

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8

Separation efficiency

Time [h] (till OTR = 4 mmol/L/h)

Oxygen transfer rate [mmol/L/h]

30

Positive control: Nitrofurantoin Negative control

A

Time till OTR = 4 mmol/L/h [h]

4

0 Positive control: Nitrofurantoin Negative control

B 8

5

Neg. control Neg. control (according to Fig. 1 B) Pos. control: Nitrofurantoin

28

26

24

4

22 1 0 0

6

12

18

24

30

36

42

2

3 4 Inoculum [%]

5

6

Time [h] Fig. 2. OTRs over time for biological replicates of the original Ames RAMOS test conducted with a preculture on complex medium as shown in Fig. 1A, generated over an experimental period of 10 months. The principle of the Ames RAMOS test conducted with precultures on complex medium is shown for a positive (0.021 μmol/L nitrofurantoin in DMSO) and negative control (DMSO) (A). Since only the OTR rise above 4 mmol/L/h is relevant for the test, the remainder of the OTR curves can be neglected (indicated by gray color). The test was conducted 8 times only for a negative control, and 8 times for both a negative and positive control (biological replicates) (B). Cultivation conditions for the Ames RAMOS test as shown in Fig. 1A: S. typhimurium TA 100, 37 °C, 250 rpm, 50 mm shaking diameter, filling volume 20 mL (250 mL flasks); preculture in RAMOS device: complex medium containing 18.8 g/L nutrient broth powder (Oxoid nutrient broth No. 2), pH0 = 7.5; test culture: minimal medium containing 1 mg/L histidine, pH0 = 7.0, OD0 = 45 ± 5 FAU (resulting in different inoculum volumina, as explained in Fig. 3).

two time spans is defined as separation efficiency. After glucose is consumed, both OTR curves decrease before rising again due to the consumption of citrate (Kauffmann et al., 2019). Since only the OTR rise until 4 mmol/L/h is relevant, the remainder of the OTR curves can be neglected (indicated by gray color in Fig. 2A). Results of this original Ames RAMOS test have been recorded over a period of 10 months. As it can be seen in Fig. 2B, the original Ames RAMOS test shows significant variability in the time point of the OTR rise due to the growth of revertants for both negative and positive controls. This leads to an unacceptable variability in mutagenicity evaluation. The negative control reaches the threshold of 4 mmol/L/h between 26 and 30 h while the positive control reaches the threshold between 22 and 25 h. This variability of the results initiated a structured investigation of the parameters responsible. The inoculum volume in the original Ames RAMOS test varies as an inherent consequence of varying ODs at the end of the preculture on complex medium (see Fig. 1A) and a constant starting OD of 45 FAU (for strain TA 100) for the test culture. Since the inoculum volume is a varying factor, the dependency of the original Ames RAMOS test results on the inoculum volume was investigated. The time until the OTR reaches the threshold of 4 mmol/L/h is plotted against its inoculum volume in Fig. 3. The filled symbols indicate the results also shown in Fig. 2B. The open symbols represent results obtained by using an extra-diluted preculture to obtain a broader inoculum volume range. This allows a deeper insight into the effects of the inoculum volume on the original Ames RAMOS test results. A dependence of the time until the OTR reaches the 4 mmol/L/h threshold value on the inoculum volume can be recognized from Fig. 3. The more preculture is used for inoculation of the test culture, the earlier the OTR rises and reaches the 4 mmol/L/h threshold value. This trend is visible for the positive and negative control, as indicated by the two trend lines. If negative and positive controls would vary to the same extent, the time difference between them could still be used to reliably determine mutagenicity.

Fig. 3. Variation of the original Ames RAMOS test results conducted as shown in Fig. 1A in dependency of the inoculum volume. The empty symbols represent results obtained by using an extra-diluted preculture (see Fig. 1B). Cultivation conditions for the Ames RAMOS test as shown in Fig. 1A: S. typhimurium TA 100, 37 °C, 250 rpm, 50 mm shaking diameter, filling volume 20 mL (250 mL flasks); preculture in RAMOS device: complex medium containing 18.8 g/L nutrient broth powder (Oxoid nutrient broth No. 2), pH0 = 7.5; test culture in RAMOS device: minimal medium containing 1 mg/L histidine, pH0 = 7.0, OD0 = 45 ± 5 FAU.

However, it can be deduced from the two trend lines that the more inoculum is transferred, in total not only more mutants arise, but also the bigger is the time difference between the controls. Thus, the system becomes more sensitive as minor changes can be differentiated by this bigger time difference. Reasons for the dependency of the original Ames RAMOS test results on the inoculum volume have to be found. 3.2. Determination of initial histidine concentration in the test culture of the original Ames RAMOS test with a preculture on complex medium Since the complex preculture medium contains histidine, histidine is also transferred into the test culture of the original Ames RAMOS test proportional to the inoculum volume. Thus, it was determined how much histidine is present in total at the beginning of the test culture of the original Ames RAMOS test. In order to investigate the histidine transfer of the different inoculum volume more closely, the metabolically accessible histidine concentration in the used lot of Oxoid nutrient broth No. 2 was investigated. The evaluation method is outlined in Supplementary file 1. The amount of metabolically accessible histidine in the complex growth medium calculates to 42.8 mg/L histidine. Due to partial consumption by the histidine auxotrophic bacteria, not all metabolically accessible histidine is still existent at the end of the preculture on complex growth medium. Thus, the histidine consumption during the preculture had to be determined. This is also outlined in Supplementary file 1. A histidine consumption of 11.75 mg/L is determined for a standard preculture of S. typhimurium TA 100 on complex medium. By subtracting the amount of consumed histidine (11.75 mg/L) from the initial amount of metabolically accessible histidine in the complex growth medium (42.80 mg/L), the amount of histidine after terminating the preculture can be calculated. Hence, after terminating the preculture, 31.05 mg/L of histidine are still present. For different inoculum volume, different amounts of histidine are transferred to the test culture of the original Ames RAMOS test. The calculated amounts of histidine at the beginning of the original Ames RAMOS test for different inoculum volume are shown in Fig. 4. Independently from the 1 mg/L histidine added to the minimal medium of the test culture, histidine is transferred with the inoculum from the preculture (31.05 mg/L). Hence, the more inoculum is transferred, the more histidine is additionally transferred. When using an inoculum

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volume of 3%, the total initial histidine amount in the original Ames RAMOS test accumulates to roughly 2 mg/L. This is two times the amount of histidine which is supposed to be in the minimal medium, according to ISO Guideline 11350 (2012) and Reifferscheid et al. (2012). The investigations above were conducted with strain TA 100. It can be assumed that the same procedure can also be applied for strain TA 98. As shown in Fig. 3, inoculum volumes of up to 3% are realistic for strain TA 100, which has a required starting OD of 45 FAU. For strain TA 98, which has a required starting OD of 180 FAU, the inoculum volume can reach even higher values of up to 6%. When using 6% inoculum volume, the total amount of metabolically accessible histidine in the test culture of the original Ames RAMOS test calculates to over 2.5 mg/L. For this study, always the same lot Oxoid nutrient broth No. 2 was used. Even if a complex ingredient is always obtained from the same manufacturer, the complex mixture of compounds, including histidine, may differ from lot to lot (Diederichs et al., 2014). Hence, also the transferred histidine can differ from lot to lot. A correlation between inoculum volume and the results of the original Ames RAMOS test could be identified. The amount of histidine transferred to the original Ames RAMOS test from the preculture to the test culture was correlated for the used complex medium (Oxoid nutrient broth No. 2). Thus, histidine transfer from the preculture to the test culture is most probably the reason for the observed variance. Since the trend depicted in Fig. 3 is not perfectly clear, further investigation is needed to confirm the statement made above. 3.3. Optimization of minimal medium for a defined preculture procedure To rationally investigate if the histidine transfer from the preculture into the test culture of the original Ames RAMOS test is a reason for the observed variance, a histidine-free preculture of histidine auxotrophic bacteria is needed. By centrifuging and washing the preculture, most of the histidine is removed. But depending on the efficiency of the washing step, some histidine might still be present. Thus, to establish welldefined conditions, a shift from the complex preculture medium (Oxoid nutrient broth No. 2) to a minimal preculture medium is required. In a minimal medium, the amount of histidine can precisely be adjusted, leading to a depletion of histidine prior to the depletion of 3.0 Initial histidine = HisMM + HisPC

Initial histidine [mg/L]

2.5

2.0

1.5

Additional histidine from preculture (HisPC)

the carbon source. Hence, the minimal medium used for the test culture of the Ames RAMOS test should also be used for the preculture. For usage of the minimal medium in the preculture, the growth of S. typhimurium TA 100 on this medium was investigated and the medium was optimized as described in Supplementary file 2. This optimized minimal medium composition is also used to conduct the following test cultures of the optimized Ames RAMOS test. For the preculture, the histidine content was adjusted to 20 mg/L for a depletion of histidine prior to preculture termination as also described in Supplementary file 2. With the optimized minimal medium composition and preculture strategy, histidine transfer from preculture to the test culture is strictly prevented. 3.4. Influence of initial histidine concentration in the test culture To rationally investigate the histidine transfer problem, an optimized Ames RAMOS test procedure without histidine transfer from the preculture was developed (see Section 3.3). Following, the initial histidine amount in the used optimized minimal medium for the test culture was varied as depicted in Fig. 1C. A total OTR curve over time for each applied histidine concentration is plotted in Supplementary file 3. For each initial histidine concentration (triplicates), the average time until the OTR reaches the threshold value of 4 mmol/L/h due to revertant growth is plotted against the initial histidine concentration as shown in Fig. 5A. As can be seen, the standard deviations of the triplicates are fairly small, indicating a reproducible test procedure, even when using independent precultures. The time until the OTR reaches 4 mmol/L/h decreases with increasing histidine concentration. The more histidine is available the more time the auxotrophic cells have to divide and mutate. For investigating the effect of initial histidine in the test culture on mutagenicity evaluation, the separation efficiency has to be considered. The separation efficiency describes the time difference between the positive and negative control of one histidine concentration. In Fig. 5B, the separation efficiency is plotted over the applied histidine concentration. It can be seen that an increasing histidine concentration leads to an increase in separation efficiency. When applying no histidine at all, the separation efficiency becomes negative, meaning that the OTR curve of the negative control increases earlier than the one for the positive control. The reason for this phenomenon is yet unclear. The results indicate that the same sample in the same concentration will be evaluated more mutagenic when more histidine was transferred from the preculture to the test culture of the original Ames RAMOS test. This can especially be problematic when a weak mutagenic compound is evaluated as it can become a false negative result. If the initial histidine concentration is increased, more cell divisions can occur increasing the probability of back-mutations. 3.5. Optimized Ames RAMOS test with defined increased initial histidine concentration

1.0 Histidine in minimal medium according to ISO 11350 (HisMM)

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Inoculum [%] Fig. 4. Total histidine concentration at the beginning of the original Ames RAMOS test culture after a preculture on complex medium (see Fig. 1A). The total histidine concentration is the sum of histidine in the minimal medium (1 mg/L) and the additional metabolically accessible histidine from the preculture (blue line): Initial His ¼ Inoculum ð%Þ  31:05mg His=L . (For interpretation of the 100 references to color in this figure legend, the reader is referred to the web version of this article.) HisMM þ HisPC ¼ 1mg His=L þ

With reference to the results from Fig. 5, it is considered whether an initial histidine concentration other than 1 mg/L should be chosen for an optimized Ames RAMOS test. By using a histidine concentration greater than 1 mg/L the separation efficiency increases, allowing a better detection of mutagenicity. As it can also be seen in Fig. 5B, the standard deviation becomes smaller with an increased initial histidine concentration. Thus, using 5 mg/L histidine instead of 1 mg/L histidine allows for an optimized Ames RAMOS test. This can be beneficial for detecting weak mutagens or mutagens which are only present in small concentrations in samples, as it is often the case for food contact materials (Rainer et al., 2019, 2018). By optimizing the Ames RAMOS test in terms of separation efficiency, these compounds could be detected more easily. In the future, even higher initial histidine concentrations should be tested to see if the separation efficiency can be increased even further. Nevertheless, even with the Ames RAMOS test which has been optimized so

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Fig. 5. Influence of different initial histidine concentrations in the optimized minimal medium on the results of the optimized Ames RAMOS test. Time until the oxygen transfer rates (OTR) of positive (1.05 μmol/L nitrofurantoin in DMSO) and negative (DMSO) controls reach the threshold value of OTR = 4 mmol/L/h (see Fig. 2) is shown for the different histidine concentrations (A). Each condition was measured in biological triplicates (three experiments with independent precultures). The separation efficiency for each histidine concentration is shown (B). Cultivation conditions: S. typhimurium TA 100, 37 °C, 250 rpm, 50 mm shaking diameter, filling volume 20 mL (250 mL flasks), pH 0 = 7.0; preculture in RAMOS device: optimized minimal medium containing 20 mg/L histidine, termination after histidine depletion; test culture in RAMOS device: optimized minimal medium containing varying histidine concentrations, OD0 = 45 ± 5 FAU.

far, a significantly more defined mutagenicity assessment can be carried out compared to the original Ames RAMOS test, as can be seen by comparing Fig. 2B and Supplementary file 4. 3.6. Influence of cultivation temperature After optimizing the preculture strategy and initial histidine concentration in the test culture, the influence of cultivation temperature on the separation efficiency was investigated using strain TA 100 and the corresponding positive control nitrofurantoin. The recommended cultivation temperature for S. typhimurium in the Ames test is 37 °C (Maron and Ames, 1983; Mortelmans and Zeiger, 2000; Reifferscheid et al., 2012). The cells have a high maximum growth rate and, hence, growth of revertants is early visible. The optimized Ames RAMOS test at 37 °C is compared to the optimized Ames RAMOS test at 30 °C as shown in Supplementary file 5. With a decreased temperature of 30 °C, the separation efficiency increases. As already discussed, a bigger separation efficiency is beneficial for mutagenicity evaluation. Therefore, the optimized Ames RAMOS test is performed at 30 °C for the rest of this study. This temperature allows an improved resolution in mutagenicity assessment and a sufficiently fast growth rate to perform the test in less than 48 h. 3.7. Generation of dose response curves with the optimized Ames RAMOS test After optimizing the Ames RAMOS test in terms of reproducibility and separation efficiency, reference compounds were assessed in multiple concentrations to obtain dose-response curves, to demonstrate the applicability of the newly developed test system. So far, the Ames RAMOS test has been performed at shake flask scale. This is not practical for determining dose-response curves at a high throughput. Therefore, the optimized Ames RAMOS test was transferred to the μRAMOS system. Fig. 6A and B shows the assessment of a dose-response curve for strain TA 100 and nitrofurantoin. Nitrofurantoin shows a mutagenic potential for strain TA 100 in the Ames fluctuation test (Zwart et al., 2018) and is used as a positive control according to ISO Guideline 11350 (2012). Fig. 6A shows the OTR curves for the growth of strain TA 100

in the optimized Ames RAMOS test with a negative control and six concentrations of nitrofurantoin. Each experimental condition was investigated six times. The corresponding standard deviations are shown as a shadow around each curve. Each experimental condition shows the typical OTR curve as explained for Fig. 2. An exception is the experiment containing the highest nitrofurantoin concentration of 2.1 μmol/L. It shows an enhanced decrease of the OTR curve during histidine limitation between 6 and 18 h. This decrease is most probably due to the cytotoxicity of nitrofurantoin, as it is known to be cytotoxic in the Ames test at higher concentrations (Klobucar et al., 2013). Apart from this, the experiments behave as expected. The higher the concentration of the mutagen nitrofurantoin, the sooner the OTR reaches the threshold value of 4 mmol/L/h due to the growth of the revertants. A dose-response curve can now be generated from the results. This is shown in Fig. 6B. The inverted time until the OTR reaches the threshold value of 4 mmol/L/h is plotted against the nitrofurantoin concentration. It should be noted that the y-axis is inverted as a shorter time until reaching the 4 mmol/L/h threshold value represents a higher number of revertants. As already shown in Fig. 6A, the number of revertants increases with increasing nitrofurantoin concentration and, thus, a doseresponse relationship from approximately 30 h for the negative control to 25 h for the highest nitrofurantoin concentration of 2.1 μmol/L can be recognized. Hence, nitrofurantoin would correctly be identified as a mutagen in the optimized Ames RAMOS test. A dose response curve obtained with the Ames fluctuation test using the same test concentrations as in the Ames RAMOS test is shown in Supplementary file 6 for a first validation. The obtained dose response curve shows the same trend as for the Ames RAMOS test. Up to now, only strain TA 100 without addition of S9 was tested in this study. In order to evaluate whether the optimized Ames RAMOS test is broadly applicable, it must also be tested with strain TA 98 and for both strains with the addition of a metabolization system (S9 from rat liver). Strain TA 98 was tested with six concentrations of 2nitrofluorene, which according to literature is positive in the Ames test (Dunkel et al., 1984; Flückiger-Isler et al., 2004). Both TA 100 and TA 98 were tested with six concentrations of 2-aminoanthracene and the addition of S9, as 2-aminoanthracene is only positive in the Ames test after being activated (Ames et al., 1973b; Reifferscheid et al.,

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2012). The resulting dose-response curves are shown in Fig. 7A–C. The respective times until the OTR increasees are plotted against the applied concentrations. It must be noted that an OTR threshold value of 3.5 mmol/L/h is set for strain TA 98, since glucose is consumed more quickly due to the higher initial OD (Kauffmann et al., 2019) and, therefore, an OTR value of 4 mmol/L/h is not always achieved. Clear doseresponse relationships can be identified for all applied chemicals. The higher the test concentration, the faster an OTR increase can be detected, thus, the more revertants have developed. For strain TA 98 with the addition of 2-nitrofluorene, a maximum separation efficiency of approximately 5.5 h is achieved. For strain TA 100 and the addition of 2-aminoanthracene and S9, a separation efficiency of 4 h is achieved. For strain TA 98 and the addition of 2-aminoanthracene and S9, this value is 7 h. Dose response curves obtained with the Ames fluctuation test using the same test concentrations of 2-nitrofluorene and 2aminoanthracene as in the Ames RAMOS test are also shown in Supplementary file 6. The obtained dose response curves show the same trend as for the Ames RAMOS test. The results shown in Figs. 6 and 7 indicate that the optimized Ames RAMOS test can determine the mutagenicity of chemicals and provide quantitative information by recording dose-response curves. Of course, in future further validation is needed to confirm these results. By carrying out the test in 48-well microtiter plates, a high-throughput test is obtained. When triplicates are taken, 14 different compounds/dilutions can be tested per plate in addition to a negative and positive control. This setup is based on a single μRAMOS device. It can be parallelized and, thus, increase the throughput. The flexibility and the online monitoring allows for a high throughput that is comparable or can even exceed the traditional Ames test on agar plates and the Ames fluctuation test. A benefit of the presented approach here is the reduced hands on time due to the time resolved mutagenicity detection as a manual dividing of the cultures into different wells is not necessary. This leads to a reduced workload in comparison to the Ames fluctuation test. Therefore, the Ames RAMOS test can compete with other in vitro test systems, as for example the umu test, in terms of throughput and workload. The umu genotoxicity test applies genetically modified strains of the original Ames test to detect DNA damage via a colorometic read-out. This test system can be conducted in a well plate format and is very rapid as it delivers a readout within 6 h after the exposure (McDaniels et al., 1990).

3.8. Significance of the results for the established Ames test systems Even though this study deals with the optimization of the Ames RAMOS test, its findings and optimization steps are also relevant for the established Ames test systems, especially the Ames fluctuation test. Here, too, a defined starting OD is required by the ISO Guideline 11350 (2012) and, thus, the inoculum volume varies. Furthermore, a complex medium as described in ISO Guideline 11350 (2012) is used for the preculture. This leads to similar additional histidine amounts transferred from the preculture into the test culture as in the original Ames RAMOS test. Accordingly, the test results can also vary in dependence of the inoculum volume. Depending on the inoculum volume, the negative control of the Ames fluctuation test can be valid (1 to 10 reverted wells out of 48 wells as described in ISO Guideline 11350 (2012)) or not. Furthermore, weak mutagenic compounds can be assessed as mutagenic or non-mutagenic, depending on the test procedure. A positive control is valid between 25 and 48 reverted wells, which is a large bandwidth (ISO 11350, 2012) by itself. If little histidine transfer from preculture takes place, the positive control may still be at the lower end of the validity range, while the weak mutagen cannot be evaluated correctly and behaves like a negative control. Difficulties in detecting weak mutagens are frequently described in literature (Flückiger-Isler et al., 2004; Kim and Margolin, 1999; Leme et al., 2012; Reifferscheid et al., 2012). Until now, no relation between histidine transfer from the preculture and varying test results has been shown for the established Ames test systems. But the possible influence of histidine on the Ames test has been discussed in literature. Maron and Ames (1983) as well as Mortelmans and Zeiger (2000) state that the histidine content has to be kept constant in the test culture of the Ames test, because spontaneous revertants are a function of histidine concentration. Verhagen et al. (1994), and Nylund and Einisto (1992) also investigated the influence of the initial histidine concentration on spontaneous revertants in the Ames test on agar plates and the Ames fluctuation test and came to the conclusion that the number of spontaneous revertants increases with an increasing amount of initial histidine. Both studies did not focus on induced mutations. Gatehouse (1987) reviewed the influence of the initial histidine concentration in terms of spontaneous and induced mutations and concluded that the qualitative Ames test results

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of varying histidine amounts is well known for proteinaceous biological samples (Gatehouse, 1987; Maron and Ames, 1983; Nylund and Einisto, 1992; Verhagen et al., 1994) and, hence, also environmental samples, but not for the inoculum volume in the Ames fluctuation test.

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2-aminoantracene [µmol/L] Fig. 7. Dose response curves generated with the optimized Ames RAMOS test. For S. typhimurium TA 100 with the addition of S9, a dose response curve using 2aminoanthracene was generated (A). For S. typhimurium TA 98, a dose response curve using 2-nitrofluorene without the addition of S9 (B) and a dose response curve using 2aminoanthracene with the addition of S9 (C) was generated. Each concentration was measured in triplicates, except the data points indicated with a gray asterisk (duplicates). For each concentration, the average time to reach the OTR threshold value of 4 mmol/L/h (TA 100) or 3.5 mmol/L/h (TA 98) is plotted. Cultivation conditions: S. typhimurium TA 100, pH0 = 7.0, 30 °C; preculture in RAMOS device: 250 rpm, 50 mm shaking diameter, filling volume 20 mL (250 mL flasks), optimized minimal medium containing 20 mg/L histidine, termination after histidine depletion; test culture in μRAMOS device: 700 rpm, 3 mm shaking diameter, filling volume 2.4 mL (3.6 mL well volume), optimized minimal medium containing 5 mg/L histidine, OD0 = 45 ± 5 FAU (TA 100) or 180 ± 10 FAU (TA 98).

Within this study, the reproducibility of the original Ames RAMOS test, which had been introduced by Kauffmann et al. (2019), was optimized. Initially, a strong variability of the test results could be observed. The larger the inoculum from the preculture to reach the required initial OD, the more revertants were observed. The difference between negative and positive control, thus the separation efficiency, also increased with increasing inoculum volume. As a result, depending on the amount of inoculum, the same sample was estimated to be more or less mutagenic. The problem identified holds also true for the Ames fluctuation test. In this study, the reason for the influence of the inoculum volume on the test results was identified to be the histidine transfer. The preculture of the original Ames RAMOS test (Fig. 1A), as well as the Ames fluctuation test, is performed on complex medium. The initial histidine content in the complex medium is not completely consumed and is, therefore, transferred to the test culture. Depending on the histidine transfer, a greater or smaller separation efficiency occurs between a negative control and a sample. This results in the fact that depending on the inoculum volume, particularly weak mutagenic compounds could be classified as non-mutagenic or mutagenic and, thus, leading to false negative results. By optimization of the minimal medium used for the Ames RAMOS test, a preculture with complete consumption of histidine could be achieved. A fixed initial histidine concentration of 5 mg/L was specified for the optimized Ames RAMOS test. This should allow the detection of weak mutagenic compounds. In order to further increase the separation efficiency (sensitivity) of the optimized Ames RAMOS test, the cultivation temperature was reduced from 37 to 30 °C. The results obtained in this study should also be used to optimize the Ames fluctuation test in terms of reproducibility and ability of detecting weak mutagenic compounds. A further standardization of the Ames fluctuation test is necessary to achieve an increased reproducibility. Also, the use of complex medium components should be avoided. The optimized minimal medium developed in this study should also be used for the Ames fluctuation test. The optimized Ames RAMOS test was finally performed in a μRAMOS system in 48-well microtiter plates and a clear dose-response relationship was found for all mutagenic reference compounds. The optimized Ames RAMOS test, therefore, allows a reproducible and rapid mutagenicity testing in a high-throughput procedure. In comparison to the Ames test on agar plates and the Ames fluctuation test, the use of 48well microtiter plates saves material (Kauffmann et al., 2019). The newly developed test can also be performed with considerably less effort. Manual evaluation of the test results is no longer necessary. A computer calculates the OTR values online. The dilution step as necessary in the Ames fluctuation test is also eliminated. In future, additional chemicals and environmental samples will be tested with the optimized Ames RAMOS test to further validate the test and determine its specificity and sensitivity. The testing of gases will also be considered in the future as many samples to be tested are gaseous (Araki et al., 1994). Gases can be introduced in a controlled manner through the gas supply system of the RAMOS technique. Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2020.137168. Funding

would remain the same as long as a stable (non-degradable, nonactivable) mutagenic compound would be used. Of course, non-stable mutagens also exist and have to be taken into account. A falsifying effect

This work was supported in part by the DBU (Deutsche Bundesstiftung Umwelt, grant number AZ 32654).

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