# Modification of the microstructure of the CERN-CLEAR-VHEE beam at the picosecond scale modifies ZFE morphogenesis but has no impact on hydrogen peroxide production

Houda Kacem, Louis Kunz, Pierre Korysko, Jonathan Ollivier, Pelagia Tsoutsou, Adrien Martinotti, Vilde Rieker, Joseph Bateman, Wilfrid Farabolini, Gérard Baldacchino, et al.

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Houda Kacem, Louis Kunz, Pierre Korysko, Jonathan Ollivier, Pelagia Tsoutsou, et al. Modification of the microstructure of the CERN-CLEAR-VHEE beam at the picosecond scale modifies ZFE morphogenesis but has no impact on hydrogen peroxide production. Radiotherapy & Oncology, 2025, 209, pp.110942. 10.1016/J.RADONC.2025.110942. cea-05098677

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Original Article

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# Modification of the microstructure of the CERN-CLEAR-VHEE beam at the picosecond scale modifies ZFE morphogenesis but has no impact on hydrogen peroxide production

Houda Kacem<sup>a,b,c</sup>, Louis Kunz<sup>a,b</sup>, Pierre Korysko<sup>d,e</sup>, Jonathan Ollivier<sup>a,b,c</sup>, Pelagia Tsoutsou<sup>a,b</sup>, Adrien Martinotti<sup>c</sup>, Vilde Rieker<sup>d,f</sup>, Joseph Bateman<sup>d,e</sup>, Wilfrid Farabolini<sup>d</sup>, Gérard Baldacchino<sup>g,h</sup>, Billy W. Loo Jr.<sup>i,j</sup>, Charles L. Limoli<sup>k</sup>, Manjit Dosanjh<sup>d,e</sup>, Roberto Corsini<sup>d</sup>, Marie-Catherine Vozenin<sup>a,b,c,*</sup>

ARTICLE INFO

Keywords:

FLASH sparing effect
Very High Energy Electron (VHEE)
Zebrafish embryo (ZFE) model
Temporal structure
Production

ABSTRACT

**Background:** FLASH radiotherapy has emerged as a promising advancement in radiation oncology, demonstrating the potential to minimize normal tissue toxicity while preserving tumoricidal efficacy. However, the precise beam parameters required for clinical translation remain to be fully defined.

**Methods:** To optimize beam parameters for clinical application, we employed Very High Energy Electrons (VHEE) at the CLEAR facility, capable of targeting deep-seated tumors. These were used alongside a FLASH-validated Intermediate Energy Electron (IIE) beam and a 160–225 keV X-ray beam, collectively delivering dose rates from 1 Gy/min to 10¹¹ Gy/s. High-throughput chemical assays investigated the radiochemical effects across this dose rate range, while zebrafish embryos provided an *in vivo* model to evaluate biological responses and developmental outcomes. This study offers the first comprehensive analysis of FLASH effects across a wide spectrum of dose rates and temporal parameters, from early physico-chemical interactions to complex biological systems.

**Results:** Data from CLEAR demonstrated that beam intensity, particularly bunch charge, is a critical determinant of the FLASH effect, and uncovered an unforeseen biological response when electrons are delivered over the picosecond timescale.

**Conclusion:** Our findings suggest that scanning strategies employing high intensity beamlets may be optimal for the clinical implementation of FLASH radiotherapy. These insights are pivotal for guiding the development of future FLASH protocols in radiation oncology.

<footnote><sup>a</sup> Sector of Radiobiology Applied to Radiotherapy/Radiation Oncology Department/Geneva University Hospital, Geneva, Switzerland</footnote>

<footnote><sup>b</sup> LiRR- Laboratory of Innovation in Radiobiology Applied to Radiotherapy/Faculty of Medicine/University of Geneva, Geneva, Switzerland</footnote>

<footnote><sup>c</sup> Laboratory of Radiation Oncology/Radiation Oncology Service/Department of Oncology /CHUV, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland</footnote>

<footnote><sup>d</sup> CERN, European Organization for Nuclear Research, Meyrin, Switzerland</footnote>

<footnote><sup>e</sup> University of Oxford, Oxford, UK</footnote>

<footnote><sup>f</sup> Oslo University, Oslo, Norway</footnote>

<footnote><sup>g</sup> University of Paris-Saclay, CEA, LIDYL, 91191 Gif-sur-Yvette, France</footnote>

<footnote><sup>h</sup> CY Cergy Paris Université, CEA, LIDYL, 91191 Gif-sur-Yvette, France</footnote>

<footnote><sup>i</sup> Department of Radiation Oncology, Stanford University School of Medicine, Stanford CA, USA</footnote>

<footnote><sup>j</sup> Stanford Cancer Institute, Stanford University School of Medicine, Stanford CA, USA</footnote>

<footnote><sup>k</sup> Department of Radiation Oncology, University of California, Irvine, CA, USA</footnote>

<footnote>* Corresponding author at: Sector of Radiobiology Applied to Radiotherapy/Radiation Oncology Department/Geneva University Hospital, Geneva, Switzerland. E-mail address: marie-catherine.vozenin@hug.ch (M.-C. Vozenin).</footnote>
## Introduction

Radiotherapy at ultra-high dose rate (UHDR), also known as FLASH radiotherapy, is currently seen as one of the most promising innovations in radiation oncology [1]. Experimental evidence from various preclinical models indicated that shortening radiation exposure times below 200 ms can protect normal tissues while remaining efficient against tumors, a phenomenon referred to as the FLASH effect. Recent studies have investigated the biological basis involved in normal tissue sparing [2,3]. Data reveal that the pathological response typically activated in normal tissue by radiotherapy at standard dose rates (1–10 Gy/min) is not triggered by FLASH [2,3]. Low levels of inflammation and protection of normal cells, including vascular, epithelial, mesenchymal and stem cells are consistently found after FLASH exposure whereas tumor cells are killed equally [3]. More recent studies suggest that FLASH remains effective under conditions classically known to induce tumor radiation resistance such as hypoxia [4] and immuno-depletion [5]. These findings are promising and could expand the therapeutic window of radiotherapy, offering new opportunities for cancer cure. However, several critical questions remain for safe and meaningful clinical transfer. While understanding the mechanisms responsible for the differential effect between normal versus tumor tissues would be significant, the priority to unlock the full potential of FLASH radiotherapy for the improvement of cancer treatment and patient well-being is to define the beam characteristics required to trigger the FLASH effect [3].

To date, the FLASH effect has been observed to be particle and beam-type agnostic. It has been reported with various beams, including intermediate energy pulsed electron (4–10 MeV) [6–8], photon [9,10] superconducting devices [11], and modified clinical proton beams [12–15]. Nevertheless, the precise parameters able to trigger the FLASH effect remain uncertain. While the average dose rate of 40 Gy/s is often cited as the reference FLASH dose rate, negative reports have been published using this questionable threshold [16,17] and others observed similar outcomes at 100 Gy/s [18].

FLASH has opened a new and intriguing area of research aimed at understanding how the duration of dose delivery impacts the biological response to irradiation. Key questions include understanding the temporal dynamics involved in the interaction of energetic electrons and tissues as well as the basis of the differential response triggered between healthy and tumor tissues. While time scales of elementary processes underpinning molecular physics and chemistry are well-known, our initial hypothesis was that the energy transfer from ionizing radiation to the milieu at femtosecond time scales could modify the early physico-chemical events of the water radiolysis cascade by altering the free radical cascades and recombination reactions that begin at subpicosecond scales and last over milliseconds [19]. We postulated that these events might subsequently modify the downstream biological response and that measurements in cell free aqueous based systems could be used as a surrogate to probe the physical parameters required to trigger the FLASH effect.

The current study was designed to probe this hypothesis and define the temporal parameters required to trigger the FLASH effect using CLEAR at CERN a unique 190 – 210 MeV facility. Very High Energy Electron beam (VHEE), the FLASH-validated 5.5 MeV Intermediate Energy Electron beam (IEE) eRT6/Oriatron beams were used as well as a conventional dose rate with 160 – 225 keV X-ray beam as a reference. The research delved into the physico-chemical and biological effects of these three distinct beams, each characterized by unique temporal structures and covering a broad dose rate spectrum from 1 Gy/min to 10¹¹Gy/s, using water, plasmid DNA and zebrafish embryos. Distinctive features specific to each beam-type and temporal structure were identified and showed that the intensity of the bunch delivered over picoseconds at CLEAR and of the pulse delivered over microseconds at eRT6 are critical parameters for preserving ZFE morphogenesis.

## Materials and methods

### Irradiation beam lines

In this study we used a variety of beam lines able to deliver a wide range of dose rates.

CLEAR (CERN): 190 – 210 MeV electrons were delivered at conventional 0.125 Gy/s and 0.2 Gy/s) and FLASH (10⁸ Gy/s to 10¹¹ Gy/s) dose rates as already described [20]. Chemical samples and ZFE were placed in individual PCR tubes (0.2 mL) positioned in a 3D-printed holder within a water tank. The samples were then placed in the beam using c-robot [21].

eRT6 (Oriatron, PMB/Alcen): 5.5 MeV electrons were delivered at conventional (0.1 Gy/s) and FLASH (60 Gy/s to 10⁷ Gy/s) dose rates as described before [22,23]. Chemical samples and ZFE were placed in individual Eppendorf tubes (2 mL) and positioned vertically in a water tank

CLEAR beam is made of trains, each made up of Gaussian-shaped bunches of 3 ps where bunch charge, number and spacing between bunches can be varied. The dose rate can be varied from 0.1 up to 10¹¹ Gy/s. eRT6 is made of pulses, each made up of Gaussian-shaped bunches of 4.63 ps that cannot be modified. The dose rate can be varied from 0.1 up to 10⁷ Gy/s. The historical nomenclature was retained, referring to the beam structures as 'train' for CLEAR and 'pulse' for eRT6, even though they essentially represent the macrostructure.

3) The Xrad 225CX/225 keV (Pxi Precision X-ray) and RS2000 160 keV (Rad Source Technologies) X-ray tubes were used as a reference for photon irradiation at conventional dose rates (0.037 and 0.07 Gy/s). Chemical samples and ZFE were placed in individual Eppendorf tubes (2 mL) and positioned vertically to the X-ray source.

Irradiation parameters are detailed in Supplementary Tables 1 2 & 3. Dosimetry was performed according to previously cross-validated protocols [24,25]. Both beams were linear accelerators (linacs) that have radiofrequency (RF) cavities but the thermionic injector for eRT6 did not allow to control the bunch structure so, the smallest accessible temporal resolution was the pulse while for CLEAR beam composed of trains of bunches, allowed for the smallest controllable structure to be the individual bunch.

### Water radiolysis

$G^-(H_2O_2)$: Primary yields of hydrogen peroxide.

To determine the primary yield of hydrogen peroxide produced during water radiolysis, we followed the production of H₂O₂ as a function of HO⁺ scavenger concentration (NaNO₂/NaNO₃), as described in [26]. Milli-Q water with a resistivity of 18.2 M.Ω.cm was equilibrated to 1%O₂, (Biospherix, Xvivo-system X3) and irradiated as indicated in (Table S1, S2&3a, supplementary data). Water samples were immediately probed post-irradiation with Amplex Red assay kit (Thermo Fisher). Fluorescence quantification was performed using Promega GloMax plate reader (Excitation: 520 nm; Emission: 580 – 640 nm).

### Production of hydrogen peroxide

To get closer to biological conditions, water samples, subject to various conditions known to influence radiation response in biological systems, were used. Oxygen levels, temperature, and scavengers varied according to (Supplementary Table 4), and water samples were irradiated as previously described (Supplementary Tables 1, 2 & 3). H₂O₂ analysis was conducted similarly to the earlier method.

### Simulation method

Chemsimul software was used to calculate the concentration of hydrogen peroxide produced after the homogenous phase of chemistry
and across the different dose rates. The software utilizes a set system of differential equations to solve which requires the use of known primary yields at the start of the simulation. More details can be found on the software website: https://chemsimul.dk/.

## DNA damage in pBR322 plasmid

To investigate DNA damage, pBR322 plasmid (Thermo Fisher Inc.) was purified and diluted at 40 ng/µL in deionized, RNAase and DNAase-free water (UltraPure). Plasmids were then irradiated according to the methods described previously in (Supplementary Tables 1, 2 & 3). Radiation induced modifications were resolved using Agarose Gel Electrophoresis (AGE) with 0.8 % agarose in Tris-acetate-EDTA (TAE) buffer. The compact supercoiled form and the two relaxed forms, open circular, and linear, were quantified using densitometric analysis (UVITEC Cambridge) and ImageJ software “gels” add-on. DNA damage was investigated in various environmental conditions such as different oxygen levels, scavengers, and labile iron (Fe²⁺) concentrations, as shown in (Supplementary Table 5). The McMahon model was applied to the data to describe the damage on plasmid resulting from irradiation [27]. The model helped in quantifying the rates of Single Strand Breaks (SSB) and Double Strand Breaks (DSB), denoted as βs and βD, respectively. Python code used for the fitting of the model is available at http://gitlab.unige.ch/lirr/PlasmidFitting. This allowed for a quantitative comparison of the amount of damaged plasmids by different irradiation types and in different environments.

## Zebrafish embryo irradiation

AB Wild Type ZF (Danio rerio, #1175, F7 generation, EZRC) were bred to produce zebrafish embryos at the PTZ (CHUV/UNIL, Lausanne, Switzerland). According to Swiss and European ethics regulations, no ethical approval is required for use of ZFE before 5 days of development that were considered as biodosimeters. 4 h post fertilization (hpf) ZFE were irradiated in water at 28 °C using the various beam lines as described in (Supplementary Tables 1, 2 & 3).

Radiation-induced alterations in ZFE length were measured at 5 days post-fertilization (dpf) following embryo fixation (4 % PFA) and microscopic imaging (Evos XL Core Cell Imaging System, Thermo Fisher). Analysis was conducted using ImageJ software.

The % ZFE Length Deficit is the following:

$$ \%ZFE \text{ Length Deficit} = \left( 1 - \left( \frac{l_{RT}}{\langle l_{CTRL} \rangle} \right) \right) \times 100 $$

with lRT the length of the irradiated embryo and lCTRL the average length of the control (non-irradiated) embryos.

## Statistical analysis

Statistical analyses were carried out using GraphPad Prism (v9.1) for water radiolysis, plasmids, and Zebrafish Embryos (ZFE) experiments. Fluorescence ratio of H₂O₂ production was assessed by t-test. For plasmids, the error bars correspond to standard deviation. For ZFE, data are presented as mean +/- SEM and analysis was done using Kruskal-Wallis test.

## Results

Measurement of primary yield of H₂O₂ in hypoxia (1% O₂) was done according to (Supplementary Tables 1, 2 & 3). H₂O₂ was used as a surrogate of the initial radiation-induced free radical production. For each concentration of HO· scavenger used, we plotted the G-value against the cubic root of the HO· scavenger following the Swroski's method [28] (Fig. 1a), and also considered the scavenging capacity [29] (Supplementary Figs.1a, b&c).

These values, detailed in Table 1, were similar for both FLASH and CONV exposures, consistently with the past publications (0.6–0.8 molecules/100 eV) [29–31] (Supplementary Table 6).

Then production of H₂O₂ was assessed minutes after water irradiation under atmospheric oxygen conditions (21%O₂), with or without scavengers. Interestingly, a reduction in H₂O₂ production was observed after FLASH compared to CONV exposures, independently of beam type (as shown Fig. 1b). The fluorescence ratio FLASH/CONV dose rate was 0.7 for IEE and 0.5 for VHEE. This suggests that either FLASH diminishes radical production under atmospheric conditions or that H₂O₂ is attacked by radicals in overlapping tracks. The complexity of the irradiated solution was then modified to mimic more closely the cellular biochemical conditions. For all dose rates, increasing oxygen concentration increased H₂O₂ production in a bell-shaped manner, with a low production found in hypoxic conditions, followed by a peak at physicoic levels (4%O₂), then decreasing again when approaching atmospheric conditions (Fig. 1c). This effect was independent of the dose rate, but FLASH irradiation reduced H₂O₂ production by 17 to 36% after 30Gy irradiation (Fig. 1c). The fluorescence reduction after different irradiation doses is shown in (Supplementary Fig. 2). When nitrate ions [25 mM] were added to scavenge hydrated electrons, H₂O₂ production became similar between both modalities and the fluorescence ratio reached 1. (Fig. 1d). Temperature variation, from room temperature (22 °C) to physiological human body temperature (37 – 38 °C), increased H₂O₂ production at higher temperatures, and the fluorescence ratio FLASH/CONV was enhanced at 37 °C and reached (0.77–0.80) (Supplementary Fig. 3). These results indicate a reduced production of free radicals following FLASH exposure mediated by hydrated electrons, a difference further enhanced at physiological temperature.

Simulations were then performed using Chemsimul®, a software which utilizes differential equation systems to model the radiolysis of pure water. The equation system implemented is well described in the literature [32]. Protracted concentrations, and particularly those of H₂O₂, were also calculated as a function of temperature and by considering the linear energy transfer of MeV-electrons [32]. It is important to note that the primary yields used in simulations were assumed to be constant across the various simulated dose rates. In this way, dose rate effects were only simulated from the homogeneous chemistry stage [19]. Radical reactions and their corresponding rate constants are compiled in (Supplementary Table 7). Parameters such as pulses, trains, dose rates, pulse duration and total dose were adjusted to closely match the irradiation condition of each water radiolysis experiment. The computed ratios of H₂O₂ FLASH/CONV as a function of the dose are provided in (Supplementary Table 8).

Ratios ranged from 1.51 to 1.84 and 1.17 – 1.27 for IEE and VHEE respectively. The calculated H₂O₂ concentration was higher for FLASH compared to CONV, contradicting experimental data. Simulations in presence of 25 mM nitrate ions and/or at physiological temperature conditions (37 °C) enhanced ratio up to 1.57 – 1.72 and 1.65 – 1.79 for IEE respectively as shown in (Supplementary Table 9 a, b &c). This discrepancy highlights an unexpected behavior arising under FLASH conditions [33] or the limitations of the simulations used.

To move a step closer to biological systems, plasmid irradiation was undertaken using CONV X-rays, IEE and VHEE CONV and FLASH according to (Supplementary Tables 1, 2 & 3). DNA damage was investigated under various conditions. In atmospheric conditions, with the plasmid in pure water, a dose-dependent induction of Single Strand Breaks (SSB) and Double Strand Breaks (DSB) was found but this was dose-rate independent (Fig. 2a).

The summary of the results of DSB (βD) and SSB yields (βS) calculated at atmospheric oxygen conditions produced by these systematic investigations are compiled in (Supplementary Table 10).

Adding 14 mM Dimethylsulfoxide (DMSO), a concentration 10 times lower than cellular antioxidant levels, significantly protected the plasmid from radiolytic damage, with a dose modifying factor (DMF) of
A

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B

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C

<figure><img src="image_5.png" /></figure>

D

<figure><img src="image_6.png" /></figure>

(caption on next page)
**Fig. 1.** Primary yields of hydrogen peroxide and hydrogen peroxide production after CONV and FLASH irradiation. A G-values measured in anoxic conditions (1%O₂), where Milli-Q water with a resistivity of 18.2M.Ω.cm was equilibrated to 1%O₂, the lowest level achievable with the hypoxic hood (Biospherix, Xvivo-system X3), versus cubic root of a scavenger denoted by [S] (NO₂⁻ ions for this work) obtained after irradiation with CONV and FLASH (190–220 MeV) VHEE, (5.5 MeV) IEE and CONV (225 kVp) X-rays compared to reported experiments performed with CONV (0.6 MeV) ¹³⁷Cs g-rays; [S] = [NO₂⁻] and CONV (1.2 MeV) ⁶⁰Co g-rays; [S] = [Br⁻]. B Fluorescence ratio FLASH/CONV of H₂O₂ production vs dose at 21%O₂ in pure water after irradiation with CONV and FLASH VHEE and IEE C Fluorescence intensity of H₂O₂ production at 30 Gy vs different O₂ levels after irradiation with CONV and FLASH IEE. D Fluorescence ratio FLASH/CONV of H₂O₂ production in presence or not of [NO₃⁻] = 25 mM (scavenger of aqueous electrons) after irradiation with CONV and FLASH IEE. Scavenging experiments are results of 6 experiments for IEE and duplicate experiments for CONV X-rays and VHEE (CONV and FLASH). Production of H₂O₂ at 21%O₂ are results of triplicate experiments with both beams. Production of H₂O₂ at different O₂ levels are results of duplicate experiments with IEE. [NO₂⁻] experiments were done with CONV and FLASH IEE. Each experiment had 8 points of measurements per dose.

<caption>**Table 1**</caption>

Primary yields of hydrogen peroxide. G (H₂O₂) in hypoxia (1%O₂) calculated from the Swroski [28] model for the different beam sources. The primary yields remained similar between eRT6 or CLEAR, regardless of dose rate. However, G° values at CLEAR were slightly lower than those observed at eRT6, potentially due to differences in the temporal structure of IEE and VHEE.

<table><thead><tr><th>Source</th><th>Energy</th><th>LET [keV/μm]</th><th>Dose rate [Gy/s]</th><th>[O₂] [M]</th><th>Chemical system</th><th>G°(H₂O₂) [molecules/100 eV]</th></tr></thead><tbody><tr><td>CONV VHEE</td><td>190–210 MeV</td><td>0.2</td><td>0.2<br>4.55x10⁸</td><td>1.19x10⁻⁵</td><td>[NO₂]/[NO₃]</td><td>0.6 ± 0.02<br>0.59 ± 0.02</td></tr><tr><td>FLASH VHEE</td><td></td><td></td><td>0.1</td><td></td><td></td><td>0.8 ± 0.02</td></tr><tr><td>CONV IEE</td><td>5.5 MeV</td><td>0.2</td><td>≥555</td><td></td><td></td><td>0.75 ± 0.03</td></tr><tr><td>FLASH IEE</td><td></td><td></td><td>0.037</td><td></td><td></td><td>0.78 ± 0.04</td></tr><tr><td>CONV X-rays</td><td>225 kVp</td><td>3</td><td></td><td></td><td></td><td></td></tr></tbody></table>

about 10-fold which was also dose rate independent (Fig. 2b). In addition, under hypoxic conditions a damage reduction with DMF of about 2-fold is observed, due to the radiosensitizer effect of oxygen. Under hypoxia and increased Fe²⁺ concentrations, used to mimic the tumor environment, DNA damage remained dose-rate independent (Supplementary Figs. 2c and Fig. 4).

Ultimately, we irradiated ZFE with the various beams and parameters (Fig. 3) and compared these biological results with the chemistry results described above.

ZFE irradiation was performed using X-rays, IEE and VHEE at conventional and ultra-high dose rates. X-rays at 0.037 Gy/s and IEE at 0.1 Gy/s produced similar dose-dependent defects in the morphogenesis of ZFE, with a length deficit at 10 Gy (D10) reaching 50 % with X-rays and 45 % with IEE compared to non-irradiated controls. FLASH IEE delivered in one single pulse of 10 Gy (Supplementary Tables 2 and 3), the D10 was only 30 %, showing the FLASH sparing effect and a DMF of 1.5 (Fig. 4a).

With CLEAR-VHEE, when the total dose was delivered with an average dose rate of 0.125 Gy/s versus 4.55x10⁸ Gy/s but using a similar dose in the bunch of 150 mGy, and a similar dose rate in the bunch of 5x10¹⁰ Gy/s, no differential effect was observed. The length deficit reached 40% in both cases falling between the length deficit curves obtained with X-rays/IEE CONV and IEE-FLASH (Fig. 4b). Then, the parameters were modified, as follows: a) average dose rate = 0.2 Gy/s, dose in the bunch = 20 mGy and dose rate in the bunch = 6.4x10⁹ Gy/s and b) average dose rate = 10⁹ Gy/s, dose in the bunch = 336 mGy and dose rate in the bunch = 10¹¹ Gy/s (Supplementary Tables 11 a&b). Interestingly with these parameters, a differential effect was produced, as the length deficit D10 for a) was 45 % and for b) 30 % (Fig. 4c) in the range of what was observed with the FLASH-IEE and a similar DMF of 1.5. To our knowledge, these results are the first to show that a differential effect on ZFE can be induced by variations in bunch intensity with VHEE operating at ultra-high dose rate, results that further define the FLASH sparing parameters at CLEAR.

To support the idea that intense electron delivery impacts biological outcome, we returned to the eRT6. The eRT6 is a versatile device but unlike the CLEAR, bunches cannot be adjusted and are similar when conventional and FLASH dose rates are used. However, variations of the average dose rate, dose rate in the pulse, dose in the pulse, pulse width, and frequency were implemented, while irradiating ZFE at 4hpf at a constant delivered dose of 10 Gy. A dose-rate escalation study was done with values ranging from 0.1 to 5.6x10⁶ Gy/s which is the maximum dose rate possible with eRT6 (Supplementary Table 12). These

experiments identified an average dose rate of 100 Gy/s, using 10 pulses of 1 Gy at 100 Hz with a pulse width of 1.8 µs i.e., dose rate in the pulse of 0.5x10⁶ as threshold parameters for protecting ZFE morphogenesis (Fig. 4d). Modification of the pulse width from 1.8 µs to 4 µs when a dose of 10 Gy was delivered in one, two and five pulses (Supplementary Table 13), did not modify the sparing outcome (Fig. 4e). However, consistently with the results obtained at CLEAR, modification of the pulse interval from 10 milliseconds (100 Hz) up to 10 min (Supplementary Table 14), keeping the pulse as intense as possible, did not modify the sparing outcome (Fig. 4f). These results show that neither overall time nor the pulse number (if less than 6 pulses) are driving the biological response whereas high pulse intensity at ultra-high dose rate does trigger the FLASH sparing effect with the eRT6.

When the results obtained at eRT6 and CLEAR were combined and for a dose of 10 Gy, an additional notion emerged: for a dose rate in the range of 10⁶ Gy/s, a dose of at least 1 Gy in the pulse was needed to preserve ZFE morphogenesis but at higher dose rate, in the range of 10¹¹ Gy/s, a dose in the bunch of 336 mGy was sufficient. This last result suggests that higher is the dose rate, lower the dose in the pulse/bunch can be.

Lastly, the FLASH parameters defined at CLEAR were used to measure H₂O₂ production (Supplementary Table 15) but similar ratio were found as shown (Supplementary Fig. 5). Finally, no correlation between H₂O₂ production and *in vivo* results obtained in ZFE was found (Fig. 5).

## Discussion

This study is the first comprehensive exploration investigating the impact of a large range of dose rates and various temporal parameters from early physico-chemical events to a complex biological system. Our results show that reductionist radiochemical approaches using water and plasmid are poor surrogate of the *in vivo* response that define the FLASH effect. However, they show that a different biological response can be expressed days after irradiation when electrons are delivered at the pico/nano/microsecond time scale versus minutes. Importantly, we identified that the combination of high intensity bunches and ultra-high average dose rate are the critical parameters to trigger the FLASH sparing effect at CLEAR, supported by the results obtained at eRT6 with high intensity pulses. These conclusions have significant implications for designing future FLASH devices and/or guiding research into the mechanisms of FLASH.

In the present study and aligning with historical data from the water radiolysis literature, we observed that primary radiolytic yields of
A

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B

<figure><img src="image_8.png" /></figure>

C

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

→ Supercoiled - Open Circular - Linear

<caption>**Fig. 2.** DNA damage in plasmids. A at 21%O₂ B In presence of 14 mM DMSO and C At 1%O₂ with CONV and FLASH VHEE and IEE beams. Results are from three independent experiments for all the beams.</caption>

hydrogen peroxide remain unaffected by modulation of dose rate. These findings agree with previous measurements made using pulsed electrons [34] or conventional g-ray sources [29–31]. Additional insights can be taken from our study, namely a lower primary yield of H₂O₂ was found for VHEE beams as compared with IEE beams. This phenomenon may be attributed to a hypothetical overlap of spurs at high dose rates, such as the ones provided by CLEAR. In addition, the reduced secondary production of H₂O₂ following FLASH irradiation, also suggests a possible alteration in the subsequent chemical reactions after the homogeneous phase. These results are consistent with our previous findings and align with other recent reports [33–35]. Notably, recent data from Sunnerberg et al. further support the idea that average dose rate plays a dominant role in modulating H₂O₂ yield in aqueous solutions of 4 % albumin.

Using the Amplex Red assay, they showed that as the mean dose rate increased, both H₂O₂ production and oxygen consumption decreased significantly, indicating that the average dose rate is a key determinant of radiochemical outcomes. Their study also found a strong inverse relationship between H₂O₂ yield and mean dose rate, suggesting that ultra-high dose rate irradiation alters oxidative stress dynamics that have not been validated in biological models. Yet they contradict the findings of Anderson et al. [34] and Sehested et al. [35]. These researchers reported an increased hydrogen peroxide production with pulsed electrons (> 5x10⁻⁶ Gy/s) compared to conventional g-rays. These discrepancies may stem from oxygen conditions. Anderson et al. and Sehested et al. used an oxygen concentration equal to 1x10⁻³ M, which is four times higher than the atmospheric oxygen pressure (2.5x10⁻⁴ M)
<figure><img src="image_13.png" /><figcaption>**Fig. 3.** Description of CLEAR (VHEE) and eRT6 (IEE). A Experimental set up; B Illustration example of beam structure after 10 Gy irradiation, orange and red representation are used for FLASH modality and blue is used for conventional modality. VHEE: very high energy electrons and IEE: intermediate energy electrons. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
A

<figure><img src="image_14.png" /></figure>

CONV eRT6 (0.1 Gy/s)
[Inst: 10⁴ Gy/s]

FLASH eRT6 (5.6.10⁶ Gy/s)
[Inst: 5.6.10⁶ Gy/s]

B

<figure><img src="image_15.png" /></figure>

FLASH VHEE (4.55 10⁸ Gy/s)
[Inst: 5.10¹⁰ Gy/s]

C

<figure><img src="image_16.png" /></figure>

D

<figure><img src="image_17.png" /></figure>

E

<figure><img src="image_18.png" /></figure>

F

<figure><img src="image_19.png" /></figure>

(caption on next page)
**Fig. 4.** Parametrization experiments on 4hpf ZFE. A Evaluation of length deficiency and dose response of 4hpf ZFE after exposure to X-rays (0.037 Gy/s), CONV (0.1 Gy/s) [dose rate in the bunch = 1.24x10⁶ Gy/s] and FLASH (5.6x10⁶ Gy/s) [dose rate in the bunch = 4.03x10⁸ Gy/s] IEE. B Evaluation of length deficiency and dose response of 4hpf ZFE after exposure to CONV 0.125 Gy/s and FLASH 4.88x10⁸ Gy/s VHEE with a similar dose rate in the bunch =5x10¹⁰ Gy/s. C Evaluation of length deficiency and dose response of 4hpf ZFE after exposure to VHEE delivered CONV 0.2 Gy/s and dose rate in the bunch = 6.4x10⁹ Gy/sand FLASH 10⁹ Gy/s) and dose rate in the bunch = 10¹¹ Gy/s respectively. D Evaluation of length deficiency and dose response of 4hpf ZFE after exposure to increasing dose rates E Different pulse widths and F Different frequencies. For IEE, embryos (n = 20) per dose and for VHEE irradiations, embryos (n = 8) per dose. Results are the average of 3 experiments for IEE (CONV and FLASH) and 2 experiments for CONV X-rays and VHEE (CONV and FLASH). Prior to irradiation, embryos were incubated under ambient oxygen conditions and transferred into tubes just minutes before exposure. Consequently, the dissolved oxygen level in the water corresponded to atmospheric air levels (21 %O₂).

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

**Fig. 5.** Correlation of ZFE Length deficiency and H₂O₂ production. The percentage of length deficit after 10 Gy irradiation of ZFE represented as a function of physioxic fluorescence intensity (H₂O₂ production) after 10 Gy irradiation delivered with CONV and FLASH VHEE, IEE and CONV X-rays. The dashed line represents the linear fitting. The R squared = 0.5233, suggesting a poor correlation between the two models. The filled circles indicate the different modalities of beams with their average dose rates and instantaneous dose rates in brackets.

used in our experiments. Another point of discrepancy is brought by simulation studies and related to the increased H₂O₂ production with increasing dose rate. This is inconsistent with the experimental results and suggest that other type of simulations based on molecular dynamics should also be considered [36–38]. Whereas in a previous work, we proposed that the reduced radiolytic production of H₂O₂ achieved upon FLASH irradiation, could be used as a proxy to probe FLASH beam capability and even proposed the existence of a threshold, where H₂O₂ yield ≤ 2.33 molecules/100 could be correlated with the preservation of ZFE morphogenesis [39]. The current results invalidate this idea, as the chemical measurements in this work did not correlate with biological outcomes *in vivo* using the ZFE model, even under biochemically/biologically relevant conditions (Fig. 5).

In order to investigate more complex macromolecules, pBR322 plasmids were then irradiated. Results show that DNA damage in cell-free systems was dose-rate insensitive and not modified when beam characteristics, such as the mean dose rate, the dose rate in the bunch/pulse, beam structure, particle energy were modulated. The dose-rate

independence of DNA damage in plasmids is maintained in biochemical conditions that mimic certain aspects of the tumor microenvironment (hypoxia, high Fe²⁺ levels). This highlights the insufficiency of the model to fully capture the complexity of biological *in vivo* models. Our results are consistent with earlier results by Milligan et al. [40,41] performed with dose rates ranging between 0.1 Gy/s and 1 Gy/s that included scavengers, as well as with published results produced at CLEAR [42] with dose rates above 10⁹ Gy/s. However, they contradict a recent report claiming reduced DNA damage in plasmids using UHDR electrons (46.6 Gy/s) [43]. In this later study, experimental conditions were probably improperly controlled as the plasmid was not properly purified which likely confounded their conclusions. In addition, data was fitted based on a very low number of experimental data points that further confounded a rigorous interpretation. However, in a recent study Wanstall et al. [44] irradiated pBR322 plasmids with VHEE CLEAR using two sets of beam parameters comparable to those applied in the current study involving ZFE. Interestingly, variation of the bunch dose rate and intensity induced measurable difference in single strand breaks at doses
above 90 Gy. Those results are in line with the findings of the current work. The relevance of DNA damage after FLASH vs CONV has been explored *in vivo* in mice and lead to contradictory conclusions. Fouillade et al. [45] reported less gH2AX foci after FLASH electrons (2x10² to 4x10⁷ Gy/s) in the normal lung, Levy et al. [46] showed similar levels of gH2AX foci in ID8 tumors after FLASH electrons (216 Gy/s) and CONV electrons (0.079 Gy/s). They also showed a slight decrease in repair foci after FLASH in the normal gut at early time-points, that similar residual foci by 24 h post-irradiation [46]. In an *ex-vivo* study of whole-blood peripheral blood lymphocytes (WB-PBL), using a comet assay, Cooper et al. [47] also reported reduced DNA damage after electron FLASH (2000 Gy/s) at doses higher than 20 Gy and under reduced oxygen conditions (0.25 – 0.5 %O₂). Barghough et al. [48] used a functional and quantitative DNA damage repair (DDR) assay *in vitro* and showed that electron FLASH (1x10² to 5x10⁶ Gy/s) did not alter chromosome translocations and junction structures in HEK293T cells more than CONV electron dose rates (0.08 Gy/s to 0.13 Gy/s) did. Collectively, the sum of experimental data obtained so far suggest that DNA damage is unlikely to explain the FLASH effect and results obtained with plasmids suggest that DNA damage is clearly dose rate independent at lower doses (below 10 Gy) and might be dose-rate dependent at high doses (above 20 Gy).

Our most important finding was that living organisms are sensitive to variation of electron beam intensity. At CLEAR, high intensity bunches of electrons (336 mGy at ultra-high average dose rate (average: 10⁹ and instantaneous: 10¹¹ Gy/s) spare ZFE morphogenesis while lower intensity bunches (20 mGy) at ultra-high average dose rate (average: 0,2 and Instantaneous: 6.4x10⁹ Gy/s) are more toxic to ZFE. Bunches of intermediate intensity (150 mGy) but delivered at a different average dose rate (0.125 vs 4.55x10⁸ Gy/s) lead to the same length deficit. A similar trend is observed with the eRT6 when high intensity pulses of 1 Gy or more are delivered at high average dose rate (5.6x10⁶ Gy/s) corresponding to the microsecond scale. These data indicate that intensity is a key parameter. In addition, the higher the dose rate, the lower the dose needed to trigger the FLASH sparing effect (i.e., 0.3 Gy at 10¹¹ Gy/s and 1 Gy at 10⁶ Gy/s). This is another critical observation, as it helps establish the therapeutic basis of operating FLASH in the clinical setting, where small doses per fraction remain standard of care. Interestingly, this observation is supported by results from the literature where morphogenesis sparing required doses above 30 Gy with dose rates ranging from 300 Gy/s to 7500 Gy/s in ZFE irradiated 24 hpf [49]. Furthermore, Ruan et al. [50] showed as well that the highest number of intestinal crypt cells in young mice was spared with a dose of 11.2 Gy delivered in a single FLASH pulse of 3.4 µs of electron irradiation. When the number of pulses and the time interval between two pulses (i.e., the frequency) was increased, crypt cells number was still maintained when compared to CONV [50]. The idea that high intensity pulses trigger the best dose modifying factor has also been recently confirmed by Liu et al. in mouse [51]. For a dose of 12 Gy, when they increased the dose per pulse (DPP) from 1 to 6.1 Gy with 1.5–1.7 MGy/s in the pulse and an average dose rate of 130–188 Gy/s, enhanced preservation of the number of regenerating crypts was shown. In addition, for the same when 2 pulses of 6 Gy were spaced by 8.33 milliseconds to 34 seconds at 1.7 MGy/s, crypt protection was sustained. Interestingly, these conditions remained efficient against melanoma [51]. Recently using FLASH Proton beam, Mascia et al. [52] and Sorensen et al. [53] showed that split doses with 2-minute intervals progressively reduced the normal tissue-sparing effect in a mouse skin model with 60 and 100 Gy/s. Here we report that FLASH protection was preserved when pulses were spaced up to 10 min, using an IEE, with a dose rate per pulse of 1.1 × 10⁶ Gy/s. These later results obtained in ZFE are consistent with the findings reported by Liu et al. [51] in the gut of mice where similar dose rate of 1.6 × 10⁶ Gy/s were used and with our results obtained with CLEAR beam, where the dose rate within each bunch reaches 1 × 10¹¹ Gy/s. Similarly, a recent study by Zhu et al. [54] found that mice total body irradiation with 10 Gy from a compact linear accelerator at

instantaneous dose rates above 10⁵ Gy/s significantly reduced mortality, myelosuppression, DNA damage, and apoptosis in mice. These results strongly suggest that increasing the dose rate preserves the FLASH effect. Our observations also show that the sparing of living organisms (at least ZFE) is not likely to be enhanced further at higher intensity and dose rate and suggest that the benefits of normal tissue protection reach a plateau above 10⁶ Gy/s.

## Conclusion

In conclusion, we provide the first evidence that live organisms are sensitive to variations of electron beam intensity, where compression of beam delivery time can impact biological response. While these observations are both transformational and challenging to traditional radio-biology, they do provide an evidence-based framework for the design of future FLASH clinical accelerators. Present results suggest that pencil/spot scanning techniques used to cover target volumes are feasible and FLASH compatible with VHEE and proton beams. Given the reduced doses required for FLASH sparing at high intensities and dose rates, VHEE beams seem optimally poised for clinical developments of FLASH radiotherapy.

## Fundings

Swiss National Science Foundation grant MAGIC – FNS CRS II186369 (to MCV supporting HK). Swiss Cancer Research KFS 5757–02-2023 (to MCV supporting HK and LK).

National Institutes of Health grant P01CA244091-01 (to BWL, CLL & MCV supporting JO, AM).

## CRediT authorship contribution statement

**Houda Kacem:** Writing – original draft, Methodology, Investigation.
**Louis Kunz:** Writing – original draft, Methodology, Investigation.
**Pierre Korysko:** Visualization, Validation, Methodology, Investigation.
**Jonathan Ollivier:** Methodology, Investigation.
**Peligia Tsoutsou:** Writing – original draft.
**Adrien Martinotti:** Investigation.
**Vilde Rieker:** Investigation.
**Joseph Bateman:** Investigation.
**Wilfrid Far abolini:** Investigation.
**Gérard Baldacchino:** Writing – review & editing, Formal analysis.
**Billy W. Loo:** Writing – original draft, Funding acquisition.
**Charles L. Limoli:** Writing – original draft, Supervision, Funding acquisition, Conceptualization.
**Manjit Dosanjh:** Writing – review & editing, Supervision, Conceptualization.
**Roberto Corsini:** Writing – review & editing, Supervision, Conceptualization.
**Marie- Catherine Vozenin:** Writing – original draft, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization.

## Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [BWL is a cofounder and board member of TibaRay. All remaining authors have declared no conflicts of interest.].

## Acknowledgments

The authors thank Dr C Bailat, Prs F. Amati, J. Bourhis, F. Bochud for their support. We also thank the PTZ-UNIL.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.radonc.2025.110942.
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Further reading

[55] Micke A, Lewis DF, Yu X. Multichannel film dosimetry with nonuniformity correction: multichannel film dosimetry with nonuniformity correction. Med Phys 2011;38:2523-34. https://doi.org/10.1118/1.3576105.

[56] Jaccard M, Petersson K, Buchillier T, Germond J, Durán MT, Vozenin M, et al. High dose-per-pulse electron beam dosimetry: usability and dose-rate independence of EBT3 Gafchromic films. Med Phys 2017;44:725-35. https://doi.org/10.1002/mp.12066.

[57] Farabolini, Wilfrid, Wroe, Laurence, Rieker, Vilde, Korysko, Pierre, Robertson, Cameron, Corsini, Roberto, et al. Development of reliable VHEE/FLASH passive dosimetry methods and procedures at CLEAR 26 September 2023;23:5028-5031 pages, 0.1 MB. https://doi.org/10.18429/JACOW-IPAC2023-THPM059.