Molecular Dosimetry of DNA Adducts in Mice Exposed to Ethylene Oxide

19 Ethylene oxide (EtO) is a highly reactive industrial chemical and classified as a known 20 human carcinogen with a putative mutagenic mode of action (MOA) . Its genotoxic 21 potential is primarily mediated through alkylation of DNA, resulting in the formation 22 of the mutagenic adduct O6-(2-hydroxyethyl)-2'-deoxyguanosine (O6-HE-dG). The N7-23 (2-hydroxyethyl)guanine (N7-HE-G) adduct is formed in greater abundance and is 24 generally considered to be non -mutagenic. However, dose -response relationships of 25 these DNA adducts, particularly at low inhalation exposure levels (i. e., below 3 ppm), 26 remain unknown. These data are necessary to inform the biological plausibility of 27 different statistical dose-response models that have been applied to human or animal 28 data used for cancer risk assessment. In the present study, male and female B6C3F1 29 mice were exposed to EtO (0, 0.05, 0.1, 0.5, 1, 50, 100, and 200 ppm) 6 hours/day for 30 28 consecutive days. Immediately following the last exposure, DNA was extracted from 31 lung, liver, bone marrow, and mammary gland, and further utilized to measure DNA 32 adduct levels using highly sensitive mass spectrometry platforms. N7-HE-G was 33 detected in all tissues and exposure groups, showing linear dose-response relationships 34 in the low-dose range (≤1 ppm) and increased sharply and exposure-disproportionately 35 in the high-dose range (≥50 ppm). Despite a very low limit of detection, O6-HE-dG, in 36 contrast, was not detected at exposures <50 ppm in any tissue consistent with at most a 37 shallow linear exposure response. At higher exposures (≥50 ppm), O6-HE-dG exhibited 38 a dose-response pattern of N7-HE-G. Notably the mammary gland, despite being 39 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 3 of 41 3 anatomically distant from the site of inhalation, exhibited the second -highest levels of 40 both adducts at high er doses. This study provides the first reliable quantitative dose-41 response evidence of DNA adducts in tumor target and non-target (liver) tissues across 42 a wide range of EtO exposures . The two DNA adducts differ markedly in their 43 abundance, repairability and mutagenic potential and to gether provide a molecular 44 MOA dose-response framework to inform both quantitative cancer risk assessment and 45 genotoxic hazard characterization. 46 47

Ethylene oxide; N7-(2-hydroxyethyl)guanine; O6-(2-hydroxyethyl)-2'-48 deoxyguanosine; dose-response relationship; mass spectrometry; inhalation exposure; 49 quantitative cancer risk assessment 50 51 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 4 of 41 4

52 Ethylene oxide (EtO) is a colorless, highly reactive gas with an estimated global 53 production of around 20 million tons, ranking it among the most extensively produced 54 organic chemicals worldwide (Ghosh and Godderis 2016) . Its primary industrial 55 application is as a chemical intermediate in the synthesis of products such as emulsifiers, 56 detergents, solvents, plastics, textiles, and antifreeze (Eastmond et al. 2014). In addition 57 to its industrial uses, EtO serves a critical function as a sterilizing agent for heat -58 sensitive medical and dental equipment and is employed as a fumigant for the 59 decontamination of various food products, including spices (Lynch et al. 2022) . 60 Exposures can occur to workers that make or use EtO, and to the general population 61 through its release to the environment (primarily air), and to all populations via its 62 endogenous production from ethylene (Kirman et al. 2021; Kirman et al. 2025). A mean 63 US total daily general population exposure has been reported as 3.5 ppb resulting from 64 a mean of 0.2 ppb in ambient air and 3.3 ppb attributed to endogenously generated EtO. 65 Cigarette smoke contains ethylene and EtO, and total daily exposure for approximately 66 1 pack/day smokers is equivalent to 16.6 ppb/day EtO (Kirman et al. 2025) . 67 Recommended occupational exposure limits have been set at 1 ppm since 1984 68 (ACGIH TLV 1984; OSHA PEL 1984). 69 The International Agency for Research on Cancer (IARC 2008) , United States 70 Environmental Protection Agency (USEPA 2016) and Texas Commission on 71 Environmental Quality (TCEQ 2020) have classified EtO as a known or likely human 72 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 5 of 41 5 carcinogen. These classifications are based on limited epidemiological evidence 73 primarily for lymphoid and/or breast cancers. The cancer classifications were supported 74 by evidence of rat and mouse carcinogenicity induced by chronic inhalation exposures 75 from 10 to 100 ppm, as well as associated mode of action evidence (IARC 2008; TCEQ 76 2020; USEPA 2016). 77 As a direct-acting alkylating agent, the EtO MoA is generally attributed to reactions 78 with nucleophilic sites on DNA, RNA, and proteins, leading to genotoxic and cancer 79 effects at higher exposures (Eastmond et al. 2014; Gollapudi et al. 2025; Gollapudi et 80 al. 2020; Vincent et al. 2019). Among the EtO DNA adducts, the N7-(2′-hydroxyethyl)-81 guanine (N7-HE-G) adduct is the most abundant, accounting for approximately 95% of 82 all EtO-induced DNA a dducts (Segerback 1990; Swenberg et al. 2011) . This adduct 83 forms through alkylation at the N7 position of guanine, which is the most nucleophilic 84 site among DNA bases and commonly reacts with alkylating agents (Gates et al. 2004). 85 Although N7-HE-G itself is not considered promutagenic, it is chemically unstable and 86 prone to spontaneous depurination with formation of apurinic/apyrimidinic (AP) sites. 87 Despite this theoretical mutagenic potential , repeated exposure to a EtO 100 ppm 88 tumorigenic exposure did not increase AP site DNA damage (Rusyn et al. 2005; 89 Swenberg et al. 2011) . The half -life of N7-HE-G varies depending on the tissue and 90 conditions, but it is generally short, ranging from a few hours to several days in double-91 stranded DNA (Margison et al. 1976). Given its short half-life, N7-HE-G accumulates 92 to steady-state levels typically reached after 7–10 days of repeated exposure to EtO in 93 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 6 of 41 6 rodents and as projected by PBPK modeling in humans (Filser and Klein 2018; Walker 94 et al. 2000). At steady-state levels, the number of N7-HE-G adducts formed is equal to 95 the number of adducts lost due to depurination, repair, or cell death (Pottenger et al. 96 2019). This predictable accumulation pattern makes the N7-HE-G adduct a useful 97 biomarker for internal EtO dose and exposure duration, even though it is not directly 98 mutagenic. 99 Although O 6-(2'-hydroxyethyl)-2′-deoxyguanosine (O 6-HE-dG) is a minor DNA 100 adduct formed from EtO exposure, it is considered as a critical DNA lesion due to its 101 strong mutagenic potential. O 6-HE-dG adduct is potentially mispaired with thymine 102 during DNA replication, leading to G:C →A:T transition mutations (Delaney and 103 Essigmann 2001; Mazon et al. 2010) . Unlike N7-HE-G adducts, O 6-HE-dG adducts 104 exhibit limited spontaneous depurination and are inefficiently repaired in the absence 105 of dedicated repai r pathways. Persistence of O 6-HE-dG induces activation of the 106 mismatch repair (MMR) system via recognition of O6-alkylguanine: thymine mispairs, 107 leading to cycles of futile repair and subsequent induction of apoptosis through DNA 108 damage response signaling cascades (Mazon et al. 2010) . In vivo investigations have 109 demonstrated that O 6-HE-dG adducts in rat tissues attain steady -state concentrations 110 following two weeks of EtO inhalation exposure at 300 ppm (Walker et al. 1992). The 111 relative stability and biological impact of O 6-HE-dG adducts implicate them as 112 principal contributors to EtO-mediated mutagenesis and carcinogenesis. 113 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 7 of 41 7 While dose–response relationships between EtO and DNA adduct formation have 114 been demonstrated in various in vitro and in vivo models, the precise characterization 115 of these relationships at low inhalation exposures that are relevant to the general 116 population remains limited. Several studies have shown a clear dose -dependent 117 increase in N7-HE-G adduct formation at exposure s above 1 ppm. Walker et al. 118 reported significant increases in N7-HE-G in the brain, spleen, and lung of F344 rats 119 beginning at 3 ppm of EtO exposures (Walker et al. 1990; Walker et al. 1992) . They 120 also observed that N7-HE-G adduct levels in B6C3F1 mice were approximately 2 - to 121 3-fold lower than in the same tissues of concurrently exposed rats. In contrast, O6-HE-122 dG adducts occur at nearly 300 times lower than N7-HE-G adducts in vivo, limiting the 123 ability of most studies to establish comprehensive dose–response curves for this adduct 124 across a broad range of exposures. Notably, Walker et al. only observed a significant 125 increase in O6-HE-dG adducts at EtO concentrations of 300 ppm (Walker et al. 1992). 126 Although elevated mutation frequencies in bone marrow and increased breast cancer 127 risk have been linked to EtO exposure (USEPA 2016), specific dose–response data for 128 N7-HE-G and O6-HE-dG adducts in these organs remain lacking. 129 To address these gaps, our study aims to evaluate the dose –response relationships of 130 both N7-HE-G and O 6-HE-dG adducts in male and female B6C3F1 mice , the strain 131 used in the EtO cancer bioassay (National Toxicology Program 1987). Mice aged 9–12 132 weeks were exposed to EtO via whole-body inhalation for 6 hours each day over a 28-133 day period. This exposure period was selected because both adducts reach steady state 134 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 8 of 41 8 by 4 weeks and therefore provide adduct dose -response data needed to evaluate the 135 biological plausibility of differing statistical models of cancer risk applied to human 136 studies. Eight exposure groups were tested: 0 ppm (air control), 0.05 ppm, 0.1 ppm, 0.5 137 ppm, 1 ppm, 50 ppm, 100 ppm, and 200 ppm. 138 139 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 9 of 41 9

140 Chemicals and Materials 141 Unless otherwise specified, all reagents and chemicals used in this study were 142 purchased from Sigma Aldrich (St. Louis, MO). EtO (CAS Number 75 -21-9; purity 143 99.99%) was provided by Balchem Corporation, (Green Pond, SC). The Optima LC-144 MS grade methanol (MeOH), acetonitrile (ACN), water, isopropyl alcohol (IPA) and 145 formic acid (FA) were all purchased from Thermo Fisher Scientific ( Rockford, IL). 146 NanoSep Centrifugal Devices (MWCO 3K) and stainless steel beads (5 mm) were 147 obtained from Pall Life Sciences (Port Washington, NY) and QIAGEN (Germantown, 148 MD), respectively. NucleoBond AXG 20 columns and NucleoBond buffer kit s were 149 purchased from Macherey -Nagel (Bethlehem, PA). Proteinase K was obtained from 150 VWR International, LLC (Atlanta, GA). Breathe-easier breathable tube membranes for 151 microtubes were purchased from Sigma Aldrich (product no. Z743501). Synthetic 152 standards, N7-(2-hydroxyethyl)guanine (N7-HE-G, TRC-H942200) and O6-(2-153 hydroxyethyl)-2′-deoxyguanosine (O6-HE-dG, TRC -H942020), and stable isotope 154 labeled internal standard (IST), N7-(2-hydroxyethyl)guanine-d4 (N7-HE-G-d4, TRC-155 H942202) and O6-(2-hydroxyethyl)-2′-deoxyguanosine-d4 (O6-HE-dG-d4, TRC -156 H942022) were purchased from LGC Standards (Manchester, NH). 157 Mouse Exposure Experiment with EtO 158 Mice were exposed via whole-body inhalation to filtered air (Group 1, G1), or EtO at 159 concentrations of 0.05 (G2), 0.1 (G3), 0.5 (G4), 1 (G5), 50 (G6), 100 (G7), and 200 160 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 10 of 41 10 (G8) ppm for 6 h per day over 28 consecutive days. Exposures were conducted in 1000-161 L stainless-steel and glass whole -body exposure chambers. Due to technical issues in 162 generating steady-state concentrations of G2 and G3 during the initial phase (Phase 1), 163 a second phase (Phase 2) was performed, including only G1, G2, G3, and G6 to ensure 164 exposure accuracy, as described in detail previously (Liu et al. submitted). While DNA 165 adducts were quantified for all animals, G2 and G3 data from Phase 1 were excluded 166 from the final dose -response analysis to maintain data integrity. For details regarding 167 the inhalation exposure system and environmental monitoring, refer to the Supporting 168 Information. 169 Immediately (within 2 hours) after the final exposure, the mice were sacrificed and 170 tissue samples including lung, liver, bone marrow and mammary gland were collected 171 and properly stored at –80 °C before further adduct analysis. Blood samples were also 172 collected for dose -response analyses of EtO systemic exposure , measured as N-(2-173 hydroxyethyl)-L-valine (HE -V) accumulation (Liu et al. submitted) , and for 174 genotoxicity endpoints, including micronucleus and Pig-a assays (Gollapudi 2023) . 175 Comparative analysis of the common exposure groups (G1 and G6) across Phase I and 176 II revealed consistent dose -dependent response trends with no significant interaction 177 between experimental phases, justifying the integration of data from both phases for 178 comprehensive dose -response modeling (see Supporting Information for detailed 179 statistical validation). 180 DNA Extraction 181 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 11 of 41 11 The experimental procedures for genomic DNA extraction were previously described 182 (Hsiao et al. 2022; Liu et al. 2021). In brief, tissue samples (~40 mg for lung and liver 183 samples; total collected samples for bone marrow and mammary gland tissues) were 184 homogenized in G2 solution from a NucleoBond buffer kit by a stainless steel bead via 185 the mechanical disruption of TissueLyzer (QIAGEN) for 10 min (50 Hz). Those 186 homogenized samples in G2 solution were further used for DNA purification according 187 to the manufacturer’s instruction for NucleoBond AXG 20 column sample preparation. 188 Purified DNA was reconstituted in 100 μL of water and further quantified by a 189 Nanodrop One spectrophotometer (dsDNA/slope mode, Thermo Fisher Scientific) . 190 Extracted tissue DNA samples were further aliquoted for N7-HE-G and O 6-HE-dG 191 adduct analysis. For N7-HE-G, up to 20  μg of DNA was used for adduct release. 192 However, due to limited DNA yield in bone marrow and mammary gland samples, only 193 5, 10, or 15 μg of DNA was used for some specimens from groups 6–8. Similarly, for 194 O6-HE-dG, up to 20 μg of DNA was utilized depending on the amount of DNA 195 remaining. 196 N7-HE-G Adduct Release and Purification 197 DNA solution prepared in 90  μL of water was spiked with 10  μL of N7-HE-G-d4 IST 198 (2 nM in water). N7-HE-G was released from DNA by neutral thermal hydrolysis. DNA 199 solution was incubated in hot water bath (95 °C) for 45 min. The hydrolyzed DNA was 200 filtered with a NanoSep 3 kDa filter (prewashed with water four times) at 8000 g for 201 20 min and the filtrate was further used for HPLC purification of N7-HE-G adduct. 202 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 12 of 41 12 The filtrate (80 μL) was injected into an Agilent 1200 Series UV HPLC fraction 203 collection system for purification of target DNA adducts. Analytes were sep arated by 204 reversed-phase liquid chromatography with an Atlantis C18 T3 column (150 × 4.6 mm, 205 3 μm, Waters). Detection wavelength and column temperature were set at 254 nm and 206 30 °C, respectively. Mobile phases were water with 10 mM ammonium acetate (A) and 207 methanol (B). HPLC gradient conditions were shown in Supplementary Table 1. The 208 target fraction was collected, completely dried under the breathable tube membranes in 209 the SpeedVac Vacuum concentrator before further reconstitution in 20 μL (for G1–G5 210 samples) or 200 μL (for G6–G8 samples) of water for Q Exactive HF MS and TSQ 211 Quantis QqQ MS analysis, respectively. 212 LC-ESI-MS/MS Analysis of N7-HE-G Adduct 213 ForG1–G5 samples, nanoLC -ESI-MS/MS analysis was conducted with an UltiMate 214 3000 RSLCnano system coupled to a Q Exactive HF Hybrid Quadrupole-Orbitrap mass 215 spectrometer through an EASY -Spray ion source for nanoelectrospray ionization 216 (Thermo Fisher Scientific). The N7-HE-G fraction was separated on a PepMap C18 217 analytical column (2 μm particle size, 25 cm x 75 μm i.d., catalog no. ES902). 218 Samples (6 μL) were loaded into an Acclaim PepMap C18 trapping column ( 3 μm 219 particle size, 15 cm x 75 μm i.d., catalog no. 164535) at a flow rate of 5 μL/min for 220 2.75 min using 0.1% FA in water as a loading solvent. After 2.75 min trapping time, 221 trapped analytes were eluted to an analytical column for separation. A binary solvent 222 system consisting of 0.1% FA in water (solvent A) and 0.1% FA in ACN (solvent B) 223 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 13 of 41 13 was used for LC separation at a flow rate of 300 nL/min. Targeted parallel reaction 224 monitoring (PRM) MS/MS data were acquired in the positive profile mode. For the 225 detection of N7-HE-G, an inclusion list was comprised of m/z 196.0829 (N7-HE-G) 226 and m/z 200.1080 (N7-HE-G-d4). LC gradient conditions and MS-PRM parameters are 227 shown in Supplementary Table 2. 228 For G6–G8 samples, on the other hand, LC-MS/MS analysis was performed using a 229 Vanquish UHPLC system coupled to a TSQ Quantis triple quadrupole (QqQ) mass 230 spectrometer through a Heated Electrospray Ionization probe ( HESI, Thermo Fisher 231 Scientific). The HESI ion source condition was used with default settings for a flow 232 rate at 200 μL/min. LC separation was achieved by using an ACQUITY UPLC HSS T3 233 column (1.8 μm particle, 150 x 2.1 mm i.d.). A binary solvent system consisting of 234 0.1% FA in water (sol vent A) and 0.1% FA in ACN (solvent B) was used for LC 235 separation. Targeted selected reaction monitoring (SRM) MS/MS data were acquired 236 in the positive centroid mode, transitions m/z 196.1 > m/z 152 and m/z 200.1 > m/z 152 237 for N7-HE-G and N7-HE-G-d4, respectively. LC gradient conditions and MS -SRM 238 parameters are shown in Supplementary Table 2. 239 O6-HE-dG Adduct Release, Purification, and Analysis 240 The O6-HE-dG adduct was released from DNA by enzymatic digestion. DNA solution 241 (100 μL) was added with 200 μL of 50 mM sodium phosphate/20 mM MgCl 2 buffer 242 (pH 7.2) along with 10 μL of the O6-HE-dG-d4 IST (1 nM in water) before digestion 243 by DNase I, alkaline phosphatase, and phosphodiesterase for 1 h at 37 °C with gentle 244 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 14 of 41 14 shaking (Hsiao et al. 2022; Liu et al. 2021) . The final enzymatic reaction volume was 245 410 μL. Following digestion, hydrolyzed DNA was filtered with a NanoSep 3 kDa filter 246 (prewashed with water four times) at 8000 g for 50 min to remove enzymes prior to 247 HPLC purification of O 6-HE-dG adduct. The filtrate ( 390 μL) was injected into the 248 aforementioned HPLC fraction collection system for adduct purification using the same 249 C18 column and solvent systems . HPLC gradient conditions are shown in 250 Supplementary Table 1. The target fraction was collected and completely dried before 251 further reconstitution in 15 μL of 0.1% FA for Q Exactive HF MS analysis. Additionally, 252 the amount of digested dG in each sample was quantitated by UV peak area based on 253 each freshly prepared dG calibration curve to estimate the dG amount in each sample 254 loaded into the HPLC column for adduct purification. The measured dG amount was 255 further utilized to normalize the O6-HE-dG adduct numbers. 256 The nanoLC -ESI-MS/MS analysis for O 6-HE-dG adduct was conducted with the 257 aforementioned Q Exactive HF MS system with the same trapping and analytical 258 columns. The O 6-HE-dG fraction was loaded to trapping column for 3.75 min before 259 eluting to analytical column for separation. For the detection of O6-HE-dG, an inclusion 260 list was comprised of m/z 312.1303 (O6-HE-dG) and m/z 316.1554 (O6-HE-dG-d4) for 261 PRM-MS/MS analysis. LC gradient conditions and MS-PRM parameters are shown in 262 Supplementary Table 2. 263 Data Analysis 264 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 15 of 41 15 The MS raw data was checked and analyzed with Xcalibur software (Thermo Fisher 265 Scientific). The MS response calibration curves for adduct quantitation were obtained 266 by using the integrated peak area and the known amount ratios of synthetic analytical 267 and internal standards. The quantitative analysis was done by extracting the major 268 fragment ion, as shown in Supplementary Table 2, in each corresponding PRM or 269 SRM event using Skyline v24.1.0.199 (MacLean et al. 2010). 270 Statistical Analysis 271 Dose-response regression modeling was performed using GraphPad Prism (version 272 10.2.0, San Diego, CA. For low -dose exposures (0 –1 ppm), a parametric linear 273 regression model was used to characterize the quantitative relationship between EtO 274 exposure and N7-HE-G adduct formation. Mean N7-HE-G adduct levels measured at 275 five defined EtO exposures served as the dependent variable, while EtO exposure 276 concentrations were treated as continuous independent variables. Model selection was 277 guided by visual inspection of residual plots and assessment of lack -of-fit statistics. 278 Regression outputs included slope, intercept, standard errors, and 95% confidence 279 intervals. The model demonstrated a strong goodness -of-fit (R² > 0.98). Assumptions 280 of normality and homoscedasticity were evaluated through residual diagnostics. At 281 higher EtO exposure levels (≥50 ppm), a weighted second-order polynomial regression 282 model was applied to account for the observed nonlinear dose-response relationship in 283 male and female mice. Weighted least squares estimation was used, with weights 284 calculated as the inverse variance of the mean responses, incorporating both sample 285 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 16 of 41 16 size and standard error. This approach allowed appropriate modeling of 286 heteroscedasticity across exposure groups. 287 Pairwise comparisons of N7-HE-G adduct levels were conducted using two -tailed 288 parametric Welch’s t -tests. Specifically, comparisons between each EtO exposure 289 group and the control group were performed to assess dose-related effects. Separately, 290 sex differences (male vs. female) within each exposure group were evaluated using the 291 same statistical approach to account for potential inequality of variances. For each 292 comparison, group means, standard errors, and sample sizes were used to compute t -293 statistics and corresponding p -values. A two-sided alpha level of 0.05 was applied to 294 determine statistical significance. Pairwise comparisons were conducted for descriptive 295 purposes. No adjustment for multiple comparisons was applied, as pairwise 296 comparisons were considered exploratory. 297 298 299 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 17 of 41 17

300

Development for the Detection and Quantitation of DNA Adducts 301 The primary aim of this study is to evaluate the dose response relationships of EtO in 302 the formation of DNA adducts within the mouse tissues. EtO is highly reactive and has 303 a short biological half-life, therefore, its exposure assessment relies on detecting stable 304 biomarkers in hemoglobin and DNA. It is known that EtO can directly react with DNA 305 molecules to form a variety of DNA adducts, including N7-HE-G and O6-HE-dG that 306 can be further released for analytical detection after unique experimental preparations 307 (Scheme 1). In addition, absolute quantification of DNA adducts can be achieved by 308 monitoring unique fragment ion and utilizing stable isotope -labeled internal standards 309 (Lu et al. 2022). To reveal the molecular dosimetry of DNA adducts in mice exposed 310 to EtO, we conducted a comprehensive experiment to quantify N7-HE-G and O6-HE-311 dG adducts induced directly by EtO exposure ( Figure 1 ). The nano -LC–MS/MS 312

achieved superb sensitivity, with on-column limits of detection of 30 amol and 313 3 amol for N7-HE-G and O6-HE-dG, respectively. 314 315 316 Scheme 1 . Ethylene oxide (EtO) directly reacts with DNA to form two key DNA 317 adducts. After unique cleavages by thermal hydrolysis and enzymatic digestion , N7-318 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 18 of 41 18 HE-G and O 6-HE-dG are released for quantitative analysis in mass spectrometry by 319 monitoring corresponding unique fragment ions. The red H indicates stable isotope 320 (deuterium)-labeled internal standards, N7-HE-G-d4 and O 6-HE-dG-d4, used in this 321 study for accurate absolute quantification. 322 323 Figure 1. Experimental design and sample processing workflow. B6C3F1/J mice were 324 exposed to different doses and air control via whole body inhalation for 28 consecutive 325 days. Lung, liver, bone marrow and mammary gland tissue were collected after 326 exposure for DNA extraction. Extracted DNA samples were further treated to release 327 N7-HE-G and O 6-HE-dG adducts by neutral thermal hydrolysis and enzymatic 328 digestion, respectively. Two purification steps including 3 kDa MWCO filtration and 329 HPLC fractionation were conducted to minimize the matrix effect from the complex 330 digested samples before targeted LC-MS/MS analysis of DNA adducts. 331 332 EtO-induced N7-HE-G Levels in Tissues 333 N7-HE-G adduct s in lung, the primary tissue in direct contact with inhaled 334 EtO,demonstrates the representative LC -MS/MS extracted ion chromatograms (XIC) 335 of N7-HE-G and spiked internal standard N7-HE-G-d4 in the lung tissues from male 336 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 19 of 41 19 mice exposed to air control, 1 ppm and 200 ppm of EtO (Figure 2). Endogenous N7-337 HE-G adduct s were clearly detected in control mice based on the exact mass of 338 expected fragment ion and identical retention time by comparing to its internal standard. 339 The peak area ratio of N7-HE-G over N7-HE-G-d4 was 0.037 from one of those G1 340 lung samples (Figure 2A). A significant increase in N7-HE-G generation was observed 341 with increasing EtO exposure doses. For example, the peak area ratios were 0.836 and 342 199.439 measured from the mice lung tissues after exposure with 1 ppm (G5) and 200 343 ppm (G8) of EtO exposure, respectively (Figure 2B and 2C). 344 345 346 Figure 2 . Representative LC -MS/MS PRM (A, B) and SRM (C) extracted ion 347 chromatograms of N7-HE-G (upper panel) and spiked internal standard N7-HE-G-d4 348 (lower panel) in lungs of male mice exposed to air control ( G1), 1 ppm ( G5) and 200 349 ppm ( G8) of EtO. Chemical structures of N7-HE-G and N7-HE-G-d4 and their 350 quantifying transition are annotated. The dashed line shows the major fragment ion 351 generated during MS/MS fragmentation for targeted quantification . RT, AA and NL 352 indicate retention time, peak area, and intensity, respectively. 353 354 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 20 of 41 20 To quantify EtO -induced DNA adduc ts, the number of N7-HE-G adducts was 355 determined using the peak area ratio of N7-HE-G over its internal standard, N7-HE-G-356 d4 and further normalized to the amount of DNA subjected to hydrol ysis. To more 357 clearly visualize the dose-dependent increase of N7-HE-G, two bar graphs were created 358 using quantified data from male and female mice lung tissues, separating the low (≤1 359 ppm) and high (≥50 ppm) EtO exposure groups due to the marked differ ence in N7-360 HE-G induction between these ranges (Figure 3). The quantitative profiles of N7-HE-361 G adducts in lungs of male and female mice exposed to EtO illustrated a clear dose-362 disproportionate dose-dependent response in higher EtO concentration exposures (≥50 363 ppm). Both male and female mice displayed similar increase trends. Moreover, even at 364 the lowest tested dose of 0.05 ppm EtO, a statistically significant increase in N7-HE-G 365 adduct number relative to the air control was detected. The quantified N7-HE-G adducts 366 in lung are further summarized in Table 1. These data demonstrate that N7-HE-G 367 adducts were generally proportional to external EtO exposure up to 100 ppm EtO, and 368 increased dose-disproportionately between 100 and 200 ppm EtO. 369 370 Table 1. N7-HE-G adduct numbers (10^8 NT) quantified in the lung, liver, bone marrow, and mammary gland collected from mice exposed to EtO. Values are presented as mean ± SD and rounded to an appropriate level of precision. Scientific notation is used where appropriate. Lung Liver Bone marrow Mammary gland Male Female Male Female Male Female Female Group Exposure concentration (ppm) N7-HE-G n N7-HE-G n N7-HE-G n N7-HE-G n N7-HE-G n N7-HE-G n N7-HE-G n 1 0 1.3 ± 0.7 30 1.4 ± 0.7 29 0.9 ± 0.5 30 1.0 ± 0.7 29 0.3 ± 0.2 29 0.3 ± 0.1 30 1.0 ± 0.5 22 2 0.05 2.0 ± 0.4* 10 1.6 ± 0.6 10 1.3 ± 0.2** 10 0.9 ± 0.2# 10 0.6 ± 0.2* 10 0.6 ± 0.3*** 10 1.2 ± 0.2 10 3 0.1 3.2 ± 0.7* 10 2.5 ± 0.8* 10 2.2 ± 0.8* 9 1.3 ± 0.2****,## 10 0.9 ± 0.3* 10 0.6 ± 0.1*,### 9 2.0 ± 0.2* 10 4 0.5 18 ± 4* 10 18 ± 4* 10 8.2 ± 1.7* 9 8.0 ± 1.1* 10 3.4 ± 0.7* 8 3.2 ± 0.7* 10 14 ± 3* 5 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 21 of 41 21 5 1 31 ± 2* 10 28 ± 5* 10 15 ± 2* 10 14 ± 2* 10 5.6 ± 1.0* 10 5.9 ± 1.8* 10 22 ± 3* 6 6 50 1.0 × 10^3 ± 1.2 × 10^2 * 20 8.7 × 10^2 ± 1.0 × 10^2 *,# 18 5.1 × 10^2 ± 7.3 × 10^1 * 20 4.1 × 10^2 ± 7.2 × 10^1 *,# 19 2.3 × 10^2 ± 3.8 × 10^1 * 20 2.0 × 10^2 ± 4.5 × 10^1 * 19 6.2 × 10^2 ± 1.0 × 10^2 * 16 7 100 2.3 × 10^3 ± 3.3 × 10^2 * 10 2.0 × 10^3 ± 2.6 × 10^2 *,### 10 1.2 × 10^3 ± 2.7 × 10^2 * 10 1.1 × 10^3 ± 1.8 × 10^2 * 10 5.0 × 10^2 ± 1.0 × 10^2 * 10 4.5 × 10^2 ± 7.0 × 10^1 * 10 1.4 × 10^3 ± 2.7 × 10^2 * 6 8 200 6.4 × 10^3 ± 8.7 × 10^2 * 10 6.7 × 10^3 ± 1.0 × 10^3 * 10 4.4 × 10^3 ± 8.3 × 10^2 * 10 4.8 × 10^3 ± 1.0 × 10^3 * 10 1.3 × 10^3 ± 2.8 × 10^2 * 10 1.7 × 10^3 ± 4.4 × 10^2 * 10 5.8 × 10^3 ± 1.1 × 10^3 * 5 *p <0.001; **p <0.005; ***p <0.01; ****p <0.05. Statistical significance between exposure group versus G1 control. #p <0.001; ##p <0.01; ###p <0.05. Statistical significance between male and female in each exposure group . 371 372 Figure 3. N7-HE-G adducts (per 108 NT, nucleotides) in lungs of mice exposed to low 373 (≤1 ppm, A) and high (≥50 ppm, B) EtO exposures. Each data point represents the mean 374 ± standard deviation (SD) at a given exposure level. 375 376 The dose-response plots illustrating the relationship between EtO exposure s and N7-377 HE-G adducts in male and female lung is further depicted in Figure 4. A biphasic dose-378 response relationship in N7-HE-G formation was clearly observed in both sexes 379 following EtO exposure. At low dose s (0–1 ppm), a clear linear relationship was 380 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 22 of 41 22 presented, as shown in the inset linear regression plots (R² = 0.993 for males; R² = 0.981 381 for females). At a higher concentration range (50 –200 ppm), a n increasingly steeper 382 non-linear-dose-response pattern was observed. These results demonstrated a strong 383 positive correlation between EtO exposure and N7-HE-G formation in mouse lung. 384 385 386 Figure 4. Dose-response curves of N7-HE-G adducts in lungs of male (A) and female 387 mice (B) across the full exposure range (0–200 ppm). N7-HE-G adducts (per 10^8 NT, 388 nucleotides) were quantified in lung tissue samples of male and female mice after 389 exposure to various concentrations of EtO. Each data point represents the mean ± 390 standard deviation (SD) at a given exposure . Insets show the linear regression fit for 391 the low-dose range (≤1 ppm), with corresponding equations and R² values. 392 393 N7-HE-G adducts were also analysed in liver, bone marrow and mammary gland. This 394 analysis aimed to evaluate whether EtO exposure leads to systemic distribution in DNA 395 adduct accumulation beyond the primary site of contact. The quantitative results of N7-396 HE-G adduct from those examined tissues are also summarized in Table 1. Notably, 397 all tissues exhibited the presence of adducts in the control non -exposed mice, an 398 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 23 of 41 23 observation consistent with endogenous production associated with normal body 399 metabolism (Kirman et al. 2021; Kirman et al. 2025) . Bone marrow ha d the lowest 400 endogenous N7-HE-G adduct level (0.319 and 0.305/10^8 NT for male and female 401 mice, respectively) among the four examined organs. The order of N7-HE-G adduct 402 from highest to lowest among tissues was lung, mammary gland, liver and bone marrow, 403 especially in the higher dose groups (≥0.5 ppm). The dose-response curves of N7-HE-404 G adducts presented similar patterns between tissues and male/female sex, i.e. a linear 405 increase in the low-dose range with a shallower slope (≤1 ppm) versus sublinear 406 increase with a steeper slope in the high -dose range (≥50 ppm). Moreover, statistical 407 analysis was performed to determine whether significant differences existed between 408 each EtO exposure group and the air control, and to examine potential sex -related 409 effects (Table 1 and 2). Overall, there is a significant difference between each EtO 410 exposure group and the air control, except 0.05 ppm samples from lung, liver and 411 mammary gland in female mice. Sex -dependent differences in adduct levels were 412 minimal and not statistically significant in most tissues and exposure groups. 413 O6-HE-dG Levels in Mice Exposed to High Dose of EtO 414 Figure 5 displays the representative LC-MS/MS XIC of the promutagenic O6-HE-dG 415 and spiked internal standard O6-HE-dG-d4 measured in lung from male mice exposed 416 to air control (G1) and 200 ppm of EtO (G8). Notably, endogenous O6-HE-dG adducts, 417 using up to 20 μg DNA for hydrolysis and our sample preparation methodol ogy, were 418 not detected in any tissues from air control mice (Figure 5A and Table 2). In addition, 419 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 24 of 41 24 O6-HE-dG adducts were consistently not detected in any samples from G2 (0.05 ppm) 420 to G5 (1 ppm) exposure groups, indicating that formation of this promutagenic adduct 421 requires relatively high levels of EtO exposure. 422 423 424 Figure 5. Representative LC-MS/MS PRM extracted ion chromatograms of O6-HE-dG 425 (upper panel) and spiked internal standard O6-HE-dG-d4 (lower panel) in lungs of male 426 mice exposed to air control (G1) and 200 ppm (G8) of EtO. Chemical structures of O6-427 HE-dG and O6-HE-dG-d4 and their quantifying transition are annotated. 428 429 Table 2. O6-HE-dG adduct numbers (10^8 dG) quantified in the lung, liver, bone marrow, and mammary gland collected from mice exposed to EtO. Lung Liver Bone marrow Mammary gland Male Female Male Female Male Female Female Group Exposure concentration (ppm) O6-HE-dG n O6-HE-dG n O6-HE-dG n O6-HE-dG n O6-HE-dG n O6-HE-dG n O6-HE-dG n 1 0 ND* 30 ND 30 ND 30 ND 30 ND 24 ND 23 ND 15 2 0.05 ND 10 ND 10 ND 10 ND 10 ND 10 ND 7 ND 8 3 0.1 ND 10 ND 10 ND 10 ND 10 ND 10 ND 9 ND 10 4 0.5 ND 10 ND 10 ND 10 ND 10 ND 8 ND 10 ND 4 5 1 ND 10 ND 10 ND 10 ND 10 ND 10 ND 9 ND 4 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 25 of 41 25 6 50 4.646 ± 0.891 20 5.033 ± 0.859 19 0.591 ± 0.101 20 0.500 ± 0.106** 19 1.351 ± 0.158 19 1.465 ± 0.303 19 3.052 ± 1.828 14 7 100 9.992 ± 2.253 10 11.549 ± 1.806 10 1.260 ± 0.402 10 1.197 ± 0.277 10 2.869 ± 0.448 10 3.101 ± 0.443 10 8.503 ± 4.898 4 8 200 33.750 ± 6.021 10 37.004 ± 7.061 10 4.410 ± 0.865 10 4.710 ± 1.106 10 9.000 ± 1.296 10 10.512 ± 3.169 10 26.501 ± 9.023 4 * ND: not detected (LOD = 3 amol, equivalent to <1 O6-HE-dG adduct per cell). ** Statistical significance between male and female in each exposure group, p <0.01. 430 In lung, O 6-HE-dG adducts became quantifiable starting at 50 ppm of EtO exposure, 431 with mean levels of 4.646 (male) and 5.033 (female) per 10^8 dG and increased dose-432 disproportionately at 200 ppm (33.750 and 37.004 per 10^8 dG). Notably, the analytical 433 sensitivity of our current method for O6-HE-dG detection is 3 amol, which is equivalent 434 to less than one adduct per cell. Lung exhibited the highest O 6-HE-dG formation, 435 followed by mammary gland, bone marrow, and liver. Figure 6 shows dose-response 436 curve of O6-HE-dG adducts in lungs of male and female mice following EtO exposure. 437 It is consistent with the dose-response pattern of N7-HE-G in high-dose range (≥50 438 ppm), showing a sublinear increas ing trend. Similarly, this apparent threshold and 439 increasing dose-response pattern w as also exhibited in the other examined tissues 440 (Table 2). These results collectively indicate that O 6-HE-dG formation is restricted to 441 mice exposed only to high doses of EtO. 442 443 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 26 of 41 26 444 Figure 6. Dose-response curves of O6-HE-dG adduct formation in lungs of male and 445 female mice across the full exposure range (0–200 ppm). Each data point represents the 446 mean ± standard deviation (SD) at a given exposure level. O6-HE-dG adducts were not 447 detected (ND) in the low-dose range (≤1 ppm). 448 Previous analyses have reported that N7-HE-G is formed at approximately 300 -fold 449 higher levels than O6-HE-dG in rats exposed to 300 ppm for 1 -4 weeks (Walker et al. 450 1992). Using methodology with substantively improved sensitivity for measurement of 451 O6-HE-dG and improved titration of the exposure at which O 6-HE-dG is first detected 452 (50 ppm), the ratios of N7-HE-G to O6-HE-dG formation for each tissue and respective 453 male and female gender at the 50 ppm exposure (Tables 1 and 2) are lung: 220, 173; 454 liver: 866, 816; bone marrow : 170,140; and female mammary gland : 202. The higher 455 ratio of N7-HE-G to O 6-HE-dG in the liver is likely a reflection of efficient repair of 456 O6-HE-dG adduct by methylguanine methyltransferase (MGMT) enzyme in this tissue. 457 These adduct ratios are in general agreement with those previously reported and vary 458 within an order magnitude across the examined tissues. 459 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 27 of 41 27 Strong Correlations between Tissue DNA and Blood Protein Adduct s The overall 460 protocol of the current study also included quantification of systemic EtO exposures as 461 measured by formation of EtO-induced N-(2-hydroxyethyl)-L-valine (HE-V) 462 hemoglobin adducts in blood collected from the same exposed mice used in this study 463 (Liu et al. submitted) . Interestingly, the HE-V dose-response pattern presents high 464 similarity with those of various tissue N7-HE-G patterns across all EtO exposure groups. 465 To investigate whether blood protein adducts could serve as surrogates for tissue -466 specific DNA damage, we assessed the statistical correlation between hemoglobin HE-467 V and N7-HE-G in tissues . A strong and positive linear correlation was observed 468 between blood HE -V and N7-HE-G adduct levels in lung, liver, bone marrow and 469 mammary gland (p < 0.001, Figure 7). 470 471 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 28 of 41 28 472 Figure 7. Correlation between blood HE -V levels and DNA adducts in EtO -exposed 473 mice (A: lung; B: liver; C: bone marrow; and D: mammary gland). Pearson correlation 474 between N7-HE-G adducts in lung and HE -V levels in blood across all EtO exposure 475 groups. HE-V data collected from same experimental tissues (Liu et al. submitted). 476 477 478 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 29 of 41 29

479 A key objective of the current study was to characterize the dose -response patterns of 480 EtO-induced DNA adduct formation, a molecular initiating MOA event hypothesized 481 for EtO carcinogenesis, in order to provide experimental dose-response data guiding 482 selection of a human cancer risk assessment model whose dose -response shape is 483 biologically plausible when considered against EtO’s hypothesized MoA . In order to 484 achieve this goal, sensitive methods were developed to quantify DNA adducts in bone 485 marrow, mammary tissue, lung and liver. This study demonstrates that the shape of the 486 non-mutagenic N7-HE-G adduct dose-response is linear at EtO exposures ≤1 ppm , 487 while pro-mutagenic O 6-HE-dG adducts were detected starting only at 50 ppm and 488 greater exposures. At EtO exposures ≥50 ppm, the dose-response slopes for both 489 adducts increased disproportionately with increasing EtO exposures. Importantly, in 490 addition to being the first report of describing N7-HE-G adducts at and below 1 ppm 491 EtO, the absence of detectable O6-HE-dG adducts at <1 ppm EtO is not attributable to 492 insufficient analytical sensitivity. To our knowledge, the current method provides the 493 lowest reported limit of detection for O6-HE-dG (3 amol), exceeding the sensitivity of 494 previously published LC -MS/MS assays, including those reported by Zhang et al 495 (Zhang et al. 2015). Previously reported methods achieved limits of quantitation in the 496 low-femtomole per milliliter range, whereas the present method achieves attomole -497 level sensitivity. Assuming approximately 6 pg of genomic DNA per cell, this detection 498 limit corresponds to fewer than one O6-HE-dG adduct per cell. 499 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 30 of 41 30 While N7-HE-G formation is highly responsive and abundant, its biological 500 significance as a mutagenic lesion is limited. The N7 position does not directly 501 participate in Watson -Crick base pairing, and this type of adduct is prone to 502 spontaneous depurination, potentially leading to apurinic/apyrimidinic (AP) sites rather 503 than base mispairing (Swenberg et al. 2011) . Thus, although N7-HE-G provides an 504 excellent dosimetric marker similar to HE -V in blood, it is unlikely to be a major 505 contributor to EtO-induced mutagenesis or carcinogenesis. 506 In contrast, O6-HE-dG was only detected at higher EtO exposures (≥50 ppm) (Figure 507 6 and Table 2 ). Although far less abundant than N7-HE-G, O6-HE-dG is highly 508 promutagenic due to its mispairing potential with thymine during DNA replication, 509 resulting in G:C → A:T transition mutations (Delaney and Essigmann 2001; Mazon et 510 al. 2010) . The absence of O 6-HE-dG at low -dose exposures (0.05 –1 ppm), despite 511 readily detectable N7-HE-G formation and the exquisite sensitivity of our technique, 512 underscores the disparity between internal dose measured as HE -V or N7-HE-G and 513 effective genotoxic dose. 514 The two most recent cancer risk assessments for EtO are based on the same 515 epidemiologic study of sterilant workers conducted by the National Institute of 516 Occupational Safety and Health (NIOSH) but result in cancer risk estimates with three 517 orders of magnitude difference (TCEQ 2020; USEPA 2016; Valdez-Flores et al. 2025). 518 Both TCEQ and USEPA assume a linear low -exposure extrapolation based on a 519 presumed mutagenic mode of action but apply very different dose -response models to 520 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 31 of 41 31 the NIOSH data. Both agencies applied Cox Proportional Hazards (CPH) models to the 521 NIOSH data. However, TCEQ and USEPA applied different forms of CPH model that 522 had comparable statistical significance. EPA selected a 2 -piece linear spline model 523 exhibiting an extremely steep linear slope at low exposures that shallows at higher 524 exposures. In contrast, TCEQ relied on a shallower single -slope log -linear dose -525 response model that is nearly linear across all exposures in the NIOSH study (Kirman 526 et al. 2025; TCEQ 2020; USEPA 2016; Valdez-Flores et al. 2025). 527 Although it can be argued based on statistical principles alone that the TCEQ model 528 approach is more parsimonious (simpler model), consideration of biological plausibility 529 should be the primary basis for selecting statistical models, especially when both 530 models have similar fit to the observed individual data. The EPA Science Advisory 531 Board’s review of the EPA IRIS assessment emphasized that “any model that is to be 532 considered reasonable for risk assessment must have a dose response form that is both 533 biologically plausible and consistent with the observed data.” (USEPA SAB 2015). 534 EPA carcinogen risk assessment guidelines states that “[i]f dose-response analysis 535 of nontumor key events is more informative about the carcinogenic process for an agent, 536 it can be used in lieu of, or in conjunction with , tumor incidence analysis for the 537 overall dose -response assessment. (emphasis added) ” (USEPA 2005) . Thus, 538 examination of the dose response relationships of DNA adduct formation in the current 539 study offers a biologically plausible and MOA -informed insights into which of the 540 epidemiology-based statistical dose -response models reliably predicts low -exposure 541 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 32 of 41 32 EtO cancer risks. In this same vein, a National Academy of Science review of the TCEQ 542 cancer risk approach concluded that MOA data focused on identifying the comparative 543 in vivo dose-response relationships of EtO DNA adducts to those of apical genotoxicity 544 would be valuable in guiding the selection of a statistical dose-response model that most 545 appropriately reflects a biologically plausible estimation of low -exposure EtO cancer 546 risks (National Academies of Sciences 2025). 547 Overall, the DNA adduct dose-response data are completely in consistent with the 548 postulated EtO dose-response shape derived by EPA from statistical modeling of 549 empirical epidemiology cancer data, i.e., a very steep initial increase in risk in the low 550 EtO exposure range that shallows at higher occupational exposures (USEPA 2016). In 551 contrast, the EtO DNA adduct dose -responses support a biologically-plausible 552 informed selection of a shallower single-slope linear cancer dose -response model 553 across the entire range of low to high EtO exposures (TCEQ 2020; Valdez-Flores et al. 554 2025). The implications of such dose -response model choices to EtO cancer risk 555 assessment are dramatically illustrated by the three order of magnitude lower cancer 556 risk predicted by the TCEQ (TCEQ 2020), whose cancer dose-response model assumed 557 an essentially single -slope linear dose-response based on the standard log -linear Cox 558 Proportional Hazards modeling of the same EtO occupational cancer dataset used by 559 the EPA. 560 Another key objective of the current experiment protocol was to inform the shape of 561 the EtO dose response inclusive of three levels of increasing biological MOA 562 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 33 of 41 33 information: 1) systemic EtO exposure measured by blood HE-V (Liu et al. submitted); 563 2) DNA adducts (this study); and 3) apical manifestation of cytogenetic (MN assay) 564 and mutagenic activity ( Pig-a) in blood reticulocytes and mature erythrocytes 565 (Gollapudi 2023). The shape of the dose-response for plasma HE-V exactly paralleled 566 that of cancer target organ N7-HE-G adducts (i.e., linear at ≤1 ppm, upward-bending at 567 ≥50 ppm), while apical genotoxicity was largely apparent only at 200 ppm EtO. These 568 integrated dose-response responses are consistent only with a conservative ly-assumed 569 shallow single-dose linear dose response at all three levels of biological MOA 570 considerations. 571 The EtO DNA adduct and genotoxicity findings associated with this study protocol are 572 consistent with similar higher-dose EtO induced genotoxicity dose-response patterns 573 reported in the literature, all of which lead to a conclusion that EtO is a weakly active 574 genotoxicant (Gollapudi et al. 2020; Hartwig et al. 2020; Pottenger et al. 2019) . 575 Detection of the promutagenic O6-HE-dG exclusively in the higher-dose groups aligns 576 with a hypothesis that EtO may exhibit negligible mutagenic activity attributed in part 577 to efficient low -exposure repair by O6-alkylguanine-DNA alkyltransferase (AGT ), 578 which is a mechanism that is saturated in high exposure scenarios (Jenkins et al. 2005). 579 These results indicate that the shallow nearly linear model selected by TCEQ (TCEQ 580 2020), while still representing a conservative model choice for EtO cancer risk 581 assessment, has greater biological plausibility than the 2- slope linear model with initial 582 very steep slope. 583 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 34 of 41 34 Interestingly, the mammary gland, despite being one of the most distal tissues from 584 the site of inhalation, exhibited comparable N7-HE-G adduct levels to the lung at 200 585 ppm EtO exposure, and ranked second among the four analyzed tissues at most other 586 exposure levels. A similar pattern was also observed for O6-HE-dG, with the mammary 587 gland consistently exhibiting the second highest adduct levels across all high -dose 588 exposure groups . Although these data suggest alignment with existing animal and 589 epidemiological evidence, such an interpretation is tempered by the observation that 590 the relatively crude punch-biopsy collection of mammary tissue likely resulted in 591 admixing with unknown quantities of surrounding adipose tissue. 592 The dose-response patterns for HE-V, N7- and O6-DNA adducts and genotoxicity 593 are also aligned with PBPK-modeled EtO toxicokinetic dose-response patterns in mice, 594 rats and humans (Fennell and Brown 2001) . Importantly, PBPK modeling predicted 595 EtO blood toxicokinetics were linear and with similar blood concentrations observed 596 for all species up to a 100-ppm exposure. However, and consistent with the EtO HE-V 597 data, mouse blood concentrations increased dose-disproportionately between 100 and 598 200 ppm, while rat and human concentrations exhibited continuing linear behavior at 599 exposures ≥300 ppm. The higher -exposure upward bend in mouse toxicokinetics was 600 attributed to preferential depletion of glutathione in mice resulting in reduced 601 detoxification capacity mediated by EtO glutathione conjugation (Filser and Klein 602 2018). Thus, as with the HE -V, DNA adduct and genotoxicity data, systemically-603 modeled PBPK EtO blood concentration data offer further evidence that EtO 604 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 35 of 41 35 conservatively operates by single -slope linear dose response s across a wide range of 605 EtO exposures in multiple mammalian species. 606 The strong correlation observed between blood HE -V protein adducts and tissue N7-607 HE-G adducts underscores the utility of HE -V as a minimally invasive biomarker of 608 internal EtO exposure. HE-V is an effective biomarker for EtO biomonitoring due to 609 its stability, detectability, and continuously linear dose response between low ppb 610 environmental to high ppm occupational exposures (Kirman et al. 2025). Our findings 611 clearly extend its application by demonstrating that HE -V levels not only reflect total 612 EtO burden but also correlate with N7-HE-G exposure in the tissues. This is especially 613 relevant for retrospective exposure assessment in epidemiological studies, where access 614 to tissue samples is limited and blood biomarker, HE-V, provides a practical alternative. 615 To the best of our knowledge, we showed, for the first time, significantly increased N7-616 HE-G adducts in the low exposure range ( ≤1 ppm). Regression analysis confirmed a 617 strong linear correlation between N7-HE-G formation and low-dose EtO exposure (≤1 618 ppm), and adducts were sharply increased in the high -dose exposure (Figure 4). This 619 specific pattern was observed in all analyzed tissues from both male and female mice. 620 Moreover, our data also revealed tissue -specific variations in adducts affected by EtO 621 exposure. Following inhalation exposure, EtO is readily absorbed through the 622 pulmonary alveoli and enters the systemic circulation, which facilitates delivery of 623 reactive EtO molecules to distalf liver, bone marrow, and mammary gland tissues Lung, 624 as a primary site of contact, consistently showed higher N7-HE-G and O6-HE-dG levels 625 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 36 of 41 36 compared to liver and bone marrow . Interestingly, the mammary gland, despite being 626 one of the most distal tissues from the site of inhalation, exhibited comparable N7-HE-627 G adduct levels to the lung at 200 ppm EtO exposure, and ranked second among the 628 four analyzed tissues at most other exposure levels ( Table 1). A similar pattern was 629 observed for O 6-HE-dG, with the mammary gland consistently exhibiting the second 630 highest adduct levels across all high-dose exposure groups. These findings support the 631 biological plausibility that high EtO exposures may contribute to development of both 632 lung and breast cancers, in alignment with existing animal and epidemiological 633 evidence. However, such an interpretation needs to be tempered by the observation that 634 mammary tissue was obtained by a punch biopsy of nipples that was likely admixed 635 with the surrounding adipose tissue, skin and hair. 636 637 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 37 of 41 37

638 Using stable isotope -labeled internal standards and highly specific, analytically 639 sensitive LC-MS/MS quantification, our study characterized the dose-response patterns 640 of both non-mutagenic N7-HE-G and pro-mutagenic O6-HE-dG DNA adducts across 641 multiple tissues in both sexes of mice. These analyses followed inhalation exposure to 642 a 4000 -fold range of EtO concentrations, spanning potential low parts -per-billion 643 environmental exposures up to substantially higher parts -per-million levels typical of 644 occupational settings. The results revealed a linear dose-response pattern for N7-HE-G 645 adduct formation that was highly correlated with the levels of N -(2-hydroxyethyl)-L-646 valine (HE-V), supporting the use of HE -V as a surrogate biomarker of EtO dose to 647 DNA. In contrast, the dose -response pattern for pro -mutagenic O 6-HE-dG adducts 648 provided critical molecular -level MOA data, essential for selecting a biologically 649 plausible dose-response model. Taken together, these findings support the conclusion 650 that the cancer risk to the general population from EtO exposure is conservatively 651 modeled by assuming a MOA that is characterized by a shallow single -slope linear 652 dose-response relationship. 653 654 655 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 38 of 41 38 Abbreviations 656 EtO, ethylene oxide; N7-HE-G, N7-(2-hydroxyethyl)guanine; O6-HE-dG, O6-(2-657 hydroxyethyl)-2′-deoxyguanosine; HE-V, N-(2-hydroxyethyl)-L-valine; PRM, 658 parallel reaction monitoring; HCD, higher-energy collisional dissociation; LC-MS, 659 liquid chromatography-mass spectrometry; MS/MS, tandem mass spectrometry; 660 MWCO, molecular weight cut-off; XIC, extracted ion chromatogram 661 662 Conflict of interest 663 The authors CWL, JP, JF, HZ, XW and KL declare they have no actual or potential 664 competing financial interests. Authors BBG, AAL and JS contributions were 665 supported by contracts from the American Chemistry Council to Exponent (AAL, JB) 666 or directly to BBG. 667 668

669 The research was partially supported by the UNC Superfund Research Program 670 (P42ES031007), UNC Center for Environmental Health and Susceptibility grant 671 (P30-ES779 010126), and a gift from the American Chemistry Council. We are 672 grateful to Dr. Joanna Klapacz for her contributions to the conceptualization and 673 design of the study, and to Chris Kirman for his technical review of the manuscript. 674 675 676 .CC-BY-NC-ND 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted March 27, 2026. ; https://doi.org/10.64898/2026.03.25.714191doi: bioRxiv preprint Page 39 of 41 39

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