# Toluene, styrene and methyl-ethyl-ketone inhalation: Effects on the power of cortical oscillations in rats

E. Bernal Meléndez, Thomas Venet, A. Thomas, S. Boucard, L. Guenot, L.

Merlen, S. Grossmann, E. Joubert, M. Mascherin, S. Viton, et al.

► To cite this version:

E. Bernal Meléndez, Thomas Venet, A. Thomas, S. Boucard, L. Guenot, et al., Toluene, styrene and methyl-ethyl-ketone inhalation: Effects on the power of cortical oscillations in rats. NeuroToxicology, 2025, 110, pp.31-41. 10.1016/j.neuro.2025.07.001. hal-05261580

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Toluene, styrene and methyl-ethyl-ketone inhalation: Effects on the power of cortical oscillations in rats

E. Bernal Meléndez<sup>a,*</sup>, T. Venet<sup>a,b</sup>, A. Thomas<sup>a</sup>, S. Boucard<sup>a</sup>, L. Guenot<sup>a</sup>, L. Merlen<sup>a</sup>, S. Grossmann<sup>a</sup>, E. Joubert<sup>a</sup>, M. Mascherin<sup>a</sup>, S. Viton<sup>a</sup>, L. Wathier<sup>a</sup>, F. Cosnier<sup>a</sup>, B. Pouyatos<sup>a,b</sup>

<figure><img src="image_2.png" /></figure>

ARTICLE INFO

Edited by Dr. Christoph van Thriel

Keywords:

Occupational exposure

Industrial solvents

Acute exposure

Neurotoxicity

Electrocorticography

A B S T R A C T

Exposure to volatile organic solvents, in both industrial workers and animal models, has a depressant effect on the central nervous system and alters behavior. However, the specific impact on brain activity during acute exposure has not been extensively studied. Here, we assessed how acute exposure to three common industrial solvents - toluene, styrene and methyl-ethyl-ketone (MEK) - affected the power of brain oscillations in rats. Rats (n = 14/group) were implanted with cortical electrodes which were connected to a removable headstage during exposure, to wirelessly transmit digitized electrocorticographic (ECOG) signals. Signals were continuously recorded while the rats inhaled solvent vapors (1000 ppm) for 6 h, with a 1-h control period before and after (breathing filtered air). Experiments were repeated for four successive days. In addition to brain oscillations, post-rotatory nystagmus (PRN) and sensory-motor coordination were tested following air/solvent exposure. MEK had no significant effects on the parameters tested. Styrene decreased the power of overall brain activity, but had no effect on motor activity. Toluene increased the power of fast oscillations (30–90 Hz) within minutes and further over time; concomitantly, the power of slow waves (2–12 Hz) decreased. Motor activity was slightly increased by toluene. Both toluene and styrene increased the number and duration of saccades measured by PRN. Dose-response experiments with styrene (n = 16 rats) revealed significant changes in oscillation power even at 50 ppm. These findings suggest that ECOG can be used to assess solvent effects on brain activity in real-time, surpassing the sensitivity of traditional sensorimotor tests.

1. Introduction

Volatile organic compounds (VOCs) are widely used in many in-
dustrial processes. As they emit vapors even at ambient pressure and
temperature, they can easily diffuse in indoor workshop environments,
offices, or in outdoor atmospheres. Upon inhalation, VOCs easily enter
the bloodstream gaining access to all the body's organs. As most VOCs
are lipophilic, they tend to accumulate in lipid-rich tissues such as the
central nervous system (CNS) (Bus et al., 1981; Li et al., 2021; van Thriel
and Boyes, 2022). As a result, exposure to certain VOCs causes adverse
effects on the CNS in both humans and animal models (Cruz and Bowen,
2021; van Thriel and Boyes, 2022). In animals, chronic exposure leads to
impaired learning and memory, social interaction, sensory-motor func-
tions, coordination and locomotion (Huerta-Rivas et al., 2012;

Paez-Martinez et al., 2003; Boyes et al., 2016). Among workers, the risks associated with chronic exposures to these substances include irreversible effects on cognitive performance and sensorimotor activity (Berr et al., 2010; Letellier et al., 2020; White and Proctor, 1997; Meyer-Baron et al., 2008; Keski-Santti et al., 2009), accompanied by structural and neurochemical alterations in the CNS (Keski-Santti et al., 2009; Tang et al., 2011; Visser et al., 2008; Sainio, 2015).

Several studies have shown that these symptoms are partly due to
alterations in neural transmission as a result of interactions between
VOCs and neuronal membrane receptors (Bowen et al., 2006; van Thriel
and Boyes, 2022). For instance, toluene (one of the most widely studied
industrial VOCs) disrupts the function of glutamatergic and GABAergic
receptors (Bale et al., 2005), nicotinic acetylcholine receptors (Bale
et al., 2002), dopaminergic receptors (Von Euler et al., 1987), and 5-HT3

Abbreviations: CNS, Central Nervous System; ECoG, Electrocorticography; FFT, Fast Fourier transform; MEK, methyl-ethyl-ketone; PRN, Post-rotatory nystagmus.

* Corresponding author.

E-mail address: estefania.bernal@inrs.fr (E. Bernal Meléndez).

<footnote><sup>a</sup> French Research and Safety Institute for the Prevention of Occupational Accidents and Diseases (INRS), Toxicology and Biomonitoring Division, 1 rue Morvan, Vandœuvre les Nancy F-54519, France</footnote>

<footnote><sup>b</sup> DevAH, UR3450, Université de Lorraine–Campus Brabois-Santé, Vandœuvre-lès-Nancy 54519, France</footnote>
ion channels (Lopreato et al., 2003).

Epidemiological studies conducted in industry (Herpin et al., 2008; Vouriot et al., 2005), along with animal experiments (Niklasson et al., 1993; Larsby et al., 1986; Tham et al., 1982), suggest that exposure to certain VOCs might also impair vestibular function. This effect may be linked to disruptions in cerebellar pathways.

In light of these effects, to protect workers from harmful effects on the brain and its functions, occupational exposure limits (OELs) have been established for some of these substances.

Although much less studied than the consequences of chronic exposure, a large number of industrial VOCs can also produce acute, reversible, effects on the nervous system and behavior following inhalation exposure. The acute neurotoxicity reported for these substances is characterized by reversible behavioral changes, notably demonstrated by increased response times when performing simple or complex tasks, drowsiness, loss of balance, perturbed fine motor coordination and flexes, and headaches (Gamberale et al., 1975; Evans and Balster, 1991). These effects occur rapidly and may impair workers' abilities, potentially increasing the risk of workplace accidents.

Studies of the effects of acute exposure have identified a common behavioral pattern for several compounds (for review, see Bale et al., 2011; van Thriel and Boyes, 2022). For instance, Warren et al. (2000) observed that 1,1,1-Trichloroethane increased overall activity and excitation at low concentrations, but at higher doses led to reduced activity, impaired performance, and CNS depression. The authors took this biphasic effect on behavior as support for their hypothesis that selected solvents are members of the general class of CNS depressants. In further support of this conclusion, Armenta-Reséndiz et al. (2019) recently reported that some behavioral profiles vary not only depending on the solvent to which the animals are exposed, but also depending on the solvent dose used. Furthermore, Gerasimov et al. (2002) observed alterations in dopamine levels in the prefrontal cortex of rats following inhalation exposure to toluene at 3000 ppm or isoamyl acetate at 2500 ppm for 40 min. Dopamine levels evolved over time and depended on the substance to which the animals were exposed.

Even though VOCs have long been known to have acute neurodepressant effects, the results from the literature remain somewhat difficult to interpret due to the fact that most studies are carried out using very high, almost lethal, concentrations (Armenta-Reséndiz et al., 2019; Rebert et al., 1989; Glowa, 1990), and few articles include a dose-response relationship. In addition, behavioral alterations tend to be assessed using a variety of tests and criteria, making comparisons between studies difficult. Finally, neural and behavioral data from experimental studies are acquired after the end of an inhalation exposure session, rather than during exposure (van Thriel and Boyes, 2022; Glowa, 1990). As a result, the threshold doses at which VOCs start to affect brain activity during inhalation exposure remain largely unknown, and the OELs are thus potentially overestimated.

To address this gap in our knowledge, we developed a method to objectively and quantitatively assess the acute effects of VOCs on CNS function during exposure. Electrocorticography (ECoG) was used to record and quantify cortical brain oscillations (which are remarkably preserved across mammals (Buzsaki et al., 2013)). Even though this technique is widely used to measure the effects of neuroactive drugs on electrical activity (Voiculescu et al., 2015; Duveau et al., 2016; Raith et al., 2020), it has rarely been used in the context of acute neurotoxicity research (Takeuchi and Hisanaga, 1977; Ghosh et al., 1990; Rebert et al., 1989; Matikainen et al., 1993).

Here, we used wireless ECoG to assess how acute VOC exposure affected brain oscillations. We focused on three widely used industrial VOCs with different neurotoxic properties: toluene, styrene, and methyl-ethyl-ketone (MEK).

Our goal was to identify early neurophysiological markers of acute exposure from ECoG data and to correlate them with impairments in behavioral performance. We also evaluated how the VOCs tested affected the vestibular reflex, based on post-rotatory nystagmus (PRN)

in line with our previous results (Chalansonnet et al., 2018).

## 2. Materials and methods

### 2.1. Animals

A total of 186 female Long Evans rats were used in this study (n = 144 for Experimental phase #1, and n = 42 for Experimental phase #2). Animals were purchased from Janvier Labs (Le Genest Saint Isle, France). They were 8 weeks old when they arrived at our facility. Animals were housed (two or three per cage unless specified otherwise) in ventilated cages (Techniplast), maintained in 12-h/12-h light/dark cycles and had *ad libitum* access to food and water, except during exposures. We were unable to use male rats in this study as previous work showed that their larger size and strength often caused implant failure during PRN testing, which lead to data and animal loss. Since the main goal of the present study was to validate ECoG measures by comparing them with behavioral and PRN data, only female rats were used to minimize the risk of implant loss.

### 2.2. Solvent exposure

#### 2.2.1. Solvents

Toluene [≥ 99.5 %, Analar R NORMAPUR® ACS, Reag. Ph. Eur. analytical reagent] (reference: 28676.297; Batch number: 20J264012) and MEK [ACS reagent, ≥ 99.0 %] (reference: 360473-1L Sigma-Aldrich; Batch number: SHBN5540) were purchased from VWR (Fontenay-sous-Bois, France). Styrene [99.5 %, for analysis, stabilized] (reference: 220530010; Batch number: A0423318) was purchased from Thermo Fisher Scientific (Illkirch-Graffenstaden, France).

#### 2.2.2. Exposure level measurements

Exposure parameters (temperature, humidity, airflow, pollutant concentrations, etc.) were monitored and controlled in accordance with OECD guideline 403 for chemical testing (acute inhalation toxicity). In the inhalation chambers, animals were housed in PTFE cages and separated from each other by small-mesh wire fences. The inhalation chambers were designed to sustain a dynamic and adjustable airflow (5–6 m³/h); they were maintained at a negative pressure of no more than 5 mmH₂O to prevent any leakage of the test compound. Air entering the chamber was filtered and conditioned at a target temperature of 22 ± 2 C and humidity of 55 ± 10 % (Table 1). Vapors were generated using a thermoregulated glass streamer. The solvents, delivered by pumps, were instantaneously vaporized upon contact with the heated surface and carried forward through the streamer by an additional airflow, to reach the main air inlet pipe of the exposure chambers.

Exposure levels were measured at regular intervals (three times) during the 6-h solvent exposure by collecting samples from the chamber's atmosphere using glass tubes packed with activated charcoal 18–35 mesh (VWR International S.A.S, Fontenay-sous-Bois, France). For analysis, solvents were desorbed from the activated charcoal using carbon disulfide. Internal standards (n-propyl acetate for MEK, o-xylene for toluene, and toluene for styrene) were added to samples before analysis with a Shimadzu GC-2010 Plus gas chromatograph (GC) (Shimadzu, Noisiel, France) equipped with a flame ionization detector. Samples were injected into a 30 m × 0.25 mm (1 µm film thickness) ZB-1701 column (Phenomenex), using helium as the carrier gas at a constant velocity of 40 cm/min. The oven temperature was isothermally set to 110 °C for MEK analysis, or 170 °C for toluene and styrene analysis. The injector (split mode with a split ratio of 1/20) and detector were maintained at 250 °C. The results of these analyzes were used to perform daily calibrations. During exposure, a second GC (Clarus 500, Perkin Elmer, Courtaboeuf, France) equipped with a flame ionization detector and an automatic gas sampling valve, was used to perform online concentration measurements every minute to check the stability of vapor generation throughout exposure.
<caption>**Table 1**</caption>

Exposure parameters in inhalation cells during the week of exposure to the industrial solvents (D1'-D4'), along with results of blood and brain assays for samples collected on the last day of exposure (D4') and 1 week after the exposure period (recovery). The number of cells and animals used to compute averages are indicated in parentheses. All results are expressed as mean ± SD. The exposure parameters during the week of exposure (D1'-D4') are expressed in [%] for humidity, [°C] for temperature, and [ppm] for mean concentration. Solvent levels in blood and brain assayed following exposure to the industrial solvents (D4') or one week after the last exposure (1 W) are expressed in [µg/g]. LOQ: Limit of quantification.

<table><thead><tr><th rowspan="2"></th><th rowspan="2">Humidity<br/>[%]<br/>D1' à D4'</th><th rowspan="2">Temperature<br/>[°C]</th><th rowspan="2">Concentration<br/>[ppm]</th><th colspan="2">D4' [µg/g]</th><th colspan="2">1 Week [µg/g]</th></tr><tr><th>Blood</th><th>Brain</th><th>Blood</th><th>Brain</th></tr></thead><tbody><tr><td colspan="8"><strong>Experimental phase I</strong></td></tr><tr><td>TOLUENE 1000 ppm</td><td>46 ± 4.1 (6)</td><td>23 ± 0.6 (6)</td><td>1021 ± 17 (6)</td><td>30 ± 3 (8)</td><td>59 ± 6 (8)</td><td>&lt; LOQ (8)</td><td>&lt; LOQ (8)</td></tr><tr><td>STYRENE 1000 ppm</td><td>44 ± 13.2 (4)</td><td>23 ± 0.3 (4)</td><td>1003 ± 18 (4)</td><td>53 ± 12 (8)</td><td>72 ± 33 (8)</td><td>&lt; LOQ (7)</td><td>&lt; LOQ (9)</td></tr><tr><td>MEK 1000 ppm</td><td>49 ± 3.6 (6)</td><td>23 ± 0.3 (6)</td><td>1006 ± 18 (6)</td><td>111 ± 18 (8)</td><td>100 ± 20 (8)</td><td>&lt; LOQ (8)</td><td>&lt; LOQ (8)</td></tr><tr><td colspan="8"><strong>Experimental phase II</strong></td></tr><tr><td>STYRENE 1000 ppm</td><td>49 ± 2.9 (3)</td><td>23 ± 0.2 (3)</td><td>989 ± 38 (3)</td><td>60 ± 22.3 (4)</td><td>83 ± 28.1 (7)</td><td></td><td></td></tr><tr><td>STYRENE 600 ppm</td><td>59 ± 3.8 (3)</td><td>23 ± 0.2 (3)</td><td>628 ± 21 (3)</td><td>22 ± 3.4 (4)</td><td>36 ± 5.6 (7)</td><td></td><td></td></tr><tr><td>STYRENE 200 ppm</td><td>44 ± 5.0 (3)</td><td>23 ± 0.2 (3)</td><td>209 ± 6 (3)</td><td>4 ± 1.4 (4)</td><td>7 ± 1.6 (7)</td><td></td><td></td></tr><tr><td>STYRENE 50 ppm</td><td>48 ± 3.2 (4)</td><td>23 ± 0.2 (4)</td><td>52 ± 2 (4)</td><td>1 ± 0.1 (6)</td><td>1 ± 0.3 (7)</td><td></td><td></td></tr></tbody></table>

## 2.3. Experimental procedures

Data shown were obtained in two different experimental paradigms; the timelines are shown in Fig. 1A and B, respectively. The number of animals, as well as their precise distribution, is presented in Table 2.

### 2.3.1. Experimental phase #1 (Fig. 1A)

This experimental phase aimed to evaluate the effects of three commonly used VOCs on cortical oscillatory power using ECoG, with the goal of relating neurophysiological alterations to potential functional, motor, and sensory disturbances.

For this purpose, one hundred and forty-four 8-week-old female rats were purchased from Janvier Labs (Le Genest Saint Isle, France). At 15 weeks of age, some of the animals underwent cranial surgery to implant electrodes for ECoG recordings (n = 14/solvent), or to place an anchoring screw to maintain the head during PRN measurement (n = 12/solvent). Animals were left to recover for one week, after which they were habituated daily to the experimental devices and exposure cages over a two-week period. Exposure thus started when rats were 18 weeks old. For each solvent, the exposed group included 14 rats implanted for ECoG recordings, 14 rats implanted for PRN, 14 non-implanted rats who performed behavioral tests, and 8 rats for measurement of solvent levels in blood and brain immediately after the end of exposure. Animals were placed in exposure chambers ventilated with clean air for 4 days (8 h/day, D1-D4). During this time, their ECoG signal was continuously recorded thanks to the wireless transmission platform. The following week, the same rats were exposed by inhalation for 4 days (D1'-D4') to 1000 ppm of toluene, styrene, or MEK. Daily solvent exposure lasted 6 h and was preceded and followed by a 1-h control period, during which rats breathed clean air. On the last day

<caption>**Table 2**</caption>

Number of animals and exact distribution across the different experimental phases. Numbers shown correspond to theoretical number of animals. Any discrepancies between these theoretical numbers and those in the results section are due to headset losses leading to the euthanasia of the animal (in the case of ECoG and NPR) or failed tissue collection (e.g., during blood and brain sampling at the end of exposure). ECoG: Electrocorticography; PRN: Post-Rotatory Nystagmus.

<table><thead><tr><th></th><th>ECoG</th><th>PRN</th><th>Behavior</th><th>Post-exposure<br/>solvent dosage</th><th>Total</th></tr></thead><tbody><tr><td colspan="6"><strong>Experimental phase I</strong></td></tr><tr><td>TOLUENE<br/>1000 ppm</td><td>n = 14</td><td>n = 12</td><td>n = 14</td><td>n = 8</td><td>n = 48</td></tr><tr><td>STYRENE<br/>1000 ppm</td><td>n = 14</td><td>n = 12</td><td>n = 14</td><td>n = 8</td><td>n = 48</td></tr><tr><td>MEK 1000 ppm</td><td>n = 14</td><td>n = 12</td><td>n = 14</td><td>n = 8</td><td>n = 48</td></tr><tr><td colspan="6"><strong>Experimental phase II</strong></td></tr><tr><td>STYRENE 1000,<br/>600,<br/>200, 50 ppm</td><td>n = 16</td><td></td><td></td><td>n = 28</td><td>n = 44</td></tr></tbody></table>

of tests, immediately after the end of exposure to clean air (D4) or solvent (D4'), animals performed behavioral tests (Open-Field and Locomotic: n = 14 ECoG rats; Grip Test: n = 14 non-implanted rats) or PRN measurements (n = 12). Eight animals per solvent were sequentially removed from the exposure chamber to take blood and brain samples to assay solvent levels. Brains were snap frozen in liquid nitrogen, and then stored with the blood samples at – 80 °C for subsequent chemical analysis.

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Timelines of the two experimental phases. **A.** Experimental phase #1. Rats were exposed to air (8 h/day) for 4 days (D1-D4), and then to solvent at 1000 ppm (1 h air/6 h solvent/1 h air) for 4 days (D1'-D4'). After the final exposure (D4, D4'), locomotion, sensory-motor coordination, strength, and post-rotatory nystagmus (PRN) were evaluated. Blood and brain samples were collected for solvent assay. **B.** Experimental phase #2. Rats were exposed to decreasing styrene doses (1000, 600, 200, 50 ppm) for 1 day each (1 h air/6 h styrene/1 h air), with 2–3 days of washout between exposures. Blood and brain samples were collected after 6 h of styrene exposure at each dose.</figcaption></figure>
### 2.3.2. Experimental phase #2 (Fig. 1B)

This second experimental phase was performed to assess the sensitivity of ECoG to detect alterations in brain activity during acute, single-day solvent exposures at varying concentrations. Based on findings from the experimental phase #1 (see Section 3.1), styrene was selected for further investigation in phase #2 due to its previously undocumented effects and its distinctive ECoG profile. Sixteen 15-week-old female rats were surgically implanted with ECoG electrodes, and styrene exposure began once the animals reached 18 weeks of age.

Each exposure “block” was composed of a day of exposure to clean air (8 h) under continuous ECoG monitoring, followed the next day by exposure to a defined concentration of styrene for 6 h (preceded and followed by exposure to filtered air for 1 h). Each [air/solvent] “block” was separated from the next by a washout period of 2–3 days. Four decreasing concentrations of styrene were tested in this order: 1000, 600, 200, 50 ppm. For each concentration, seven animals were sequentially removed from exposure chambers following exposure to solvent for 6 h to take blood and brain for solvent assays.

## 2.4. Quantitative electroencephalography (ECoG)

### 2.4.1. Surgical procedure and habituation

Under sterile conditions, an approximately 1.5 cm long incision was made in the skin over the vertex. The skull was cleared by cutting through the connective tissue and scraping it. A dental drill was used to drill holes bilaterally over the motor cortex (Anteroposterior (A/P): + 1.4 mm; Lateral (L): ± 3 mm) and the auditory cortex (A/P: – 3.6 mm; L: ± 6 mm), relative to Bregma. Two additional holes were drilled behind the lambdoid suture to place reference and ground screws. Stainless steel screw electrodes (length: 3.2 mm; diameter: 1.18 mm) were placed in the different holes and connected to a standard single-line 1.27-mm pin connector using insulated enameled copper wires (diameter 0.22 mm). A layer of super-bond dental cement (Super-bond universal kit, Sun Medical) was spread on the dry skull to affix the ensemble in place. The protruding heads were then embedded in Table 200 dental cement (KERR). Rats were isolated during the recovery period and monitored closely for one week. They remained individually housed thereafter to avoid deterioration of the implanted device, but were placed in transparent cages to allow visual contact with their peers. The housing environment was enriched with Sizzle Nests. After the recovery period, the animals were progressively habituated to the ECoG device and the exposure cages over 2 weeks.

### 2.4.2. ECoG recordings and signal analysis

#### 2.4.2.1. Signal acquisition.
ECoG recordings started between 20 and 23 days after electrode implantation. The ECoG recording device (W2100, Multichannel Systems) consisted of a headstage (W2100-HS8-SR) to record brain activity (sampling rate: 1000 Hz), and perform analog-to-digital conversion (16-bit), pre-amplification (Gain: 100), bandpass filtering (1–400 Hz), as well as wireless transmission. During recording sessions, the headstage was connected to a 300-mAh battery (W2100-B-300mAh-BB), attached to the implanted connector. The power source was removed at the end of recording. The complete recording apparatus weighed 13 g. ECoG activities could be continuously wirelessly recorded for 8 h from exposure chambers. At the end of each exposure day, the headstages and batteries were removed, and the batteries were recharged.

#### 2.4.2.2. Signal analyses.
Fast Fourier transforms (FFT) and Morlet wavelet calculations of cortical activities were performed using ImaGIN, an open source SPM toolbox for intracranial EEG (https://f-tract.eu/software/imagin/) written in MATLAB (The Mathworks, MA, USA).

The power changes induced by different experimental conditions were calculated as follows:

1. FFTs were used to generate power spectra over a frequency range of 1–140 Hz at a resolution of 1 Hz, using a 2-s sliding window with 90 % overlap.

2. The median of the power spectra obtained for each window was calculated over signal segments covering 10–60 min (depending on the desired temporal definition) and normalized relative to the median power spectrum of the baseline period (the hour preceding the start of exposure).

3. The calculated power changes were averaged across the frequency range expressed in frequency bands: Delta (2–4 Hz), Theta (4–8 Hz), Alpha (8–12 Hz), Beta (12–30 Hz), Low Gamma (30–50 Hz), High Gamma (50–90 Hz), Epsilon (90–140 Hz). In addition, brain oscillations were grouped into slow oscillations (2–12 Hz) and fast oscillations (30–90 Hz), corresponding to the frequency ranges most modulated by the treatments.

4. The mean power changes (calculated for the signals from the four electrodes implanted in each animal) were computed for each individual. ECoG profiles, shown in figures and tables, correspond to the average power changes observed over the first 3 days of exposure to solvent or air. The 4th day was excluded from this average because the conditions were not identical to the other days as, after 7 h, animals were removed from the cages to perform behavioral tests.

Morlet wavelets were computed over the 2–140 Hz frequency range with a frequency resolution of 2 Hz and a time resolution of 60 s. For each animal, time-frequency maps obtained during exposure to solvents were normalized relative to the maps calculated for “air” exposures.

### 2.5. Post-rotatory nystagmus (PRN)

#### 2.5.1. Surgical procedure and habituation

Under sterile conditions, an approximately 1.5 cm long incision was made in the skin over the vertex. The skull was cleared by cutting through the connective tissue and scraping it. A screw nut was placed on the skull to immobilize the animal during the PRN procedure. The nut was affixed to the bone with a thin layer of super-bond dental cement (Super-bond universal kit, Sun Medical), and then embedded in Table 2000 dental cement (KERR). Rats were isolated and monitored closely during the recovery period.

One week after surgery, the animals were progressively habituated to the PRN procedure. The first step consisted in a simple handling of the rats. Then, they were immobilized in a dark room with a soft snuggle-restraint device, leaving the head free. The final step was to immobilize the head using the screw. In total, the habituation phase lasted 10 working days before starting PRN measurements.

#### 2.5.2. PRN measurements

The test apparatus consisted of a dark circular arena (102.5 cm diameter) containing a horizontal turntable. A system to hold a rat was fixed on the turntable, centered 88 mm from the rotation axis. As a result, the rat was rotated around its own body axis (located approximately between its shoulder blades). The metal plate, cylinder, turntable, camera holder and all the other custom equipment were custom-made at our lab.

The animal's head was held in position with a screw placed on a metal bar in the center of the turntable in order to stabilize it during rotation. The head was fixed at 30°, with the nose pointing down to align the lateral semicircular canals horizontally. The turntable could rotate 360°. The maximal acceleration and deceleration were 870°/s². The rotatory stimulation lasted 40 s at 90°/s. After rotation, horizontal movement of the left pupil was recorded in the dark using a high-resolution infrared camera recording system (RK-826PCI eye tracker, 240 Hz sampling rate; ISCAN, MA, USA), placed in a holder on the wall. Nystagmus was thus recorded immediately after rotation was stopped, hence the name PRN. Measurements were taken immediately after (within 5 min of) the end of the 6-h exposure to solvent on the fourth day
of both "air" and "solvent" exposures (D4 and D4', respectively). The PRN parameters recorded were the number and the total duration of saccades.

## 2.6. Behavioral tests

Behavioral performance was assessed in tests related to locomotor activity (Open-Field), motor coordination (Locotronic), and strength (Grip Test). Devices were cleaned with a 20 % alcohol solution before every individual test to avoid potential bias due to odors and/or residues left by rats tested previously. All experiments were carried out on the fourth day of exposure, following exposure to air (D4) or to solvent (D4'), immediately after the end of the 6 h of solvent inhalation.

### 2.6.1. Locomotor activity

Locomotor activity was assessed in an automated 3D open-field platform (OF-3C, Bioseb®; length 1000 mm; width 1000 mm; height 400 mm). Rats were habituated to the test apparatus before the exposure period. During testing, rats were placed in the center of the arena, which was placed in a quiet room illuminated by red light. They were allowed to freely explore the platform for 8 min. The total distance covered (in meters) was automatically recorded.

### 2.6.2. Forelimb strength

Forelimb strength was assessed using a vertical forelimb grip strength apparatus (Ugo Basile). The animal's paws were placed on T-shaped gripping bar (height: 56 cm), which the animal naturally held onto while its tail was gently pulled down. The grip strength (maximum strength in [gF]) of the rat's forelimbs upon grip release measured by a system connected to the gripping bar were automatically recorded. Three trials were performed per animal.

### 2.6.3. Locomotor coordination

A Locotronic apparatus (Intelli-Bio®, Seichamps, France) was used to test fine motor coordination when walking. Rats crossed a 75 × 5 × 20 cm flat ladder with bars (7 mm diameter) spaced 2 cm apart. Infrared sensors above and below the bars monitored paw and tail errors. Each animal performed three trials. The total crossing time (in [s]) and total number of errors (missteps) were recorded.

## 2.7. Solvent assay in blood and brain

Solvent (toluene, styrene and MEK) concentrations in blood and brain were determined based on published protocols involving liquid/liquid extraction of the tissues (800 mL of blood or half a brain) into dichloromethane (DCM) (500 mL). Methylisobutylketone (MIBK), toluene, or n-propylbenzene were used as internal standards (10 mL, 1.7 g/L in DCM). For brain samples, a homogenate was first prepared by grinding halfbrains in 2 mL lysing matrix D tubes containing 1.4-mm ceramic beads (20 s at 4 m/s in a Fast Prep instrument, MP Biomedicals, Illkirch, France). After shaking for 30 min, samples were centrifuged for 10 min at 3000 g (or 4000 g for brain) and – 4 °C. A portion (0.3 mL) of the lower DCM layer, containing MEK, styrene or toluene, was then transferred to a vial containing a glass insert.

Samples were quantified by gas chromatography–mass spectrometry (GC–MS) on a QP2010 ultra chromatograph (Shimadzu, Noisiel, France) equipped with a split/splitless injector and an AOC20i auto-sampler and controlled by SU1 software (version 4.11). Samples were separated on an Rxi 5Sil MS capillary column (30 m × 0.25 mm, film thickness 0.25 µm with 5-m integra-guard column) (Restek, Lisses, France) using helium as the carrier gas at a linear velocity of 40 cm/s. Samples were injected in split mode with a ratio of 1/20. The oven temperature was held at 40 °C for 4 min, then increased at a rate of 15 °C/min to 235 °C, the temperature was maintained for 1 min (total duration, 18 min). The temperatures of the injection port, transfer line, and ion source were set to 240, 250 and 200 °C, respectively. The MS was operated by electron

impact ionization (70 eV) in selected ion monitoring (SIM) mode (m/z = 91 for toluene and MIBK, m/z = 104 for MEK and styrene, and m/z = 120 for n-propylbenzene). Whatever the tissue studied, in these conditions, the limits of quantification were approximately 2 µg/g for MEK and 4 µg/g for styrene and toluene, with a bias and precision (repeatability) of less than 10 %. Levels of solvent detected in blood and brain on the last day of exposure (D4') and 1 week after completing the exposure phase (recovery period) are presented in Table 1.

## 2.8. Statistical analyses

All statistical analyses were carried out using GraphPad Prism 10.2.1. On some occasions, due to interruptions in WIFI transmission, ECoG signal was lost, leading to missing data points. In these cases, repeated-measure ANOVA could not be applied. Data were thus analyzed by fitting a mixed model based on a compound symmetric covariance matrix that was adjusted using Restricted Maximum Likelihood (REML). This mixed model was applied to analyze the effects of "treatment" and "exposure duration" on "power changes". Bonferroni multiple comparison tests were then applied to compare the power changes between "solvent" and "air" recordings for all one-hour time periods. For PRN and behavioral tests, a Wilcoxon matched-pairs signed rank test was used. The threshold for statistical significance was set at $p < 0.05$.

# 3. Results

## 3.1. ECoG power changes

Exposure to 1000 ppm toluene caused a progressive increase in the global power of the oscillations from the second hour of exposure to the first hour post-exposure, peaking at around + 10 % (compared to the control period) at the end of exposure (Figs. 2C and S2). This increase in power was mainly driven by low and high Gamma oscillations (30–90 Hz) peaking at around + 30 % after 6 h (Figs. 2B, 2D, S1 and S2). A slight decrease in the power of slow oscillations (2–12 Hz; Figs. 2A and 2D) was also observed during the first two hours of exposure (Fig. S1 and S2). Following the end of toluene exposure, the power of fast oscillations remained elevated for more than an hour (Figs. 2B and 2D, S1 and S2), but power had returned to normal levels the next day (data not shown).

Exposure to 1000 ppm of styrene caused a decrease in 2–12 Hz oscillations during the first 4 h; thereafter power recovered slowly (Figs. 2A and 2D). Concomitantly, the power of 30–90 Hz oscillations increased progressively, reaching approximately + 30 % (compared to the control period) after 6 h (Figs. 2B, S1 and S2). As soon as the exposure was discontinued, the power of slow oscillations rapidly returned to baseline levels, whereas the increase in the power of high-frequency oscillations persisted for more than an hour (Figs. 2B, S1 and S2). Overall, exposure to styrene caused a significant drop in the global power of the oscillations during the first 2 h of exposure to then gradually increase and reach approximately + 20 % (compared to at the control period) after 6 h (Fig. 2C and Figs. 2D, S1 and S2). As with toluene, brain oscillation power had returned to normal the next day (i. e., 17 h after the end of exposure to the solvent; data not shown).

Unlike toluene and styrene, exposure to 1000 ppm of MEK led to no major changes in the power of brain oscillations (Figs. 2, S1 and S2).

## 3.2. Post-rotatory nystagmus

The effects of exposure to 1000 ppm of toluene, styrene, and MEK for 6 h on the number and duration of saccades are shown in Fig. 3A and B, respectively. Exposure to 1000 ppm of toluene induced a 40 % increase in the number of saccades ($W = 68.0, p = 0.0054$) and a 37 % increase in their duration ($W = 68.0, p = 0.0049$). Exposure to 1000 ppm of styrene, increased the number of saccades by 53 % ($W = 45.0,$
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** ECoG power modifications (mean ± SD) of (A) slow oscillations (2–12 Hz), (B) fast oscillations (30–90 Hz), and (C) overall signal relative to the baseline period (first hour of “air” exposure) over time. Power changes were averaged over the first three days of exposure to “air” (D1 to D3, in black) or to the solvent of interest (D1' to D3', in color). (D) Time-Frequency map of average ECoG power obtained during solvent exposure normalized against the control period of “air” exposure. The color scale indicates an increase (shades of red) or decrease (shades of blue) of power relative to “air” exposure (z-score).</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>**Fig. 3.** Number of saccades (A) and saccade duration (B) of post-rotatory nystagmus (PRN) after a 40-second stimulation at 90°/s, measured for each solvent on D4 (fourth day of air exposure, in white) and D4' (fourth day of exposure to the solvent of interest, in color). Data are presented as scatter plots and mean ± SD. \* p < 0.05, \*\* p < 0.01. A non-parametric test on paired data was performed (Wilcoxon test).</figcaption></figure>

p = 0.0039) and their duration by 87 % (W = 45.0, p = 0.0039). In contrast, exposure to 1000 ppm of MEK had no effect on the number (W = 29.0, p = 0.2178) or duration (W = 26.0, p = 0.2783) of saccades.

solvents.

### 3.3. Locomotor behavior and strength

Animals exposed to 1000 ppm of toluene for 6 h covered more distance in the open-field arena than non-exposed animals (W = 66.0, p = 0.006; Table 3), they also crossed the Locotronic device faster (W = −71.0, p = 0.010) and with fewer errors (W = −53.0, p = 0.038). No significant differences were observed following exposure to the other two solvents for any of the behavioral tests performed.

Grip strength in the exposed animals was unchanged with all three

### 3.4. Effects of different concentrations of styrene on ECoG profiles

As observed in experimental phase #1, exposure to 1000 ppm of styrene caused a rapid decrease in the power of all brain oscillation frequency bands over the first three hours. The total reduction of about 35 % after two hours indicates a strong neuro-depressive effect (Figs. 4C, S3 and S4). Slow oscillations (2–12 Hz) remained suppressed throughout the exposure period (Figs. 4A, S3 and S4), whereas a biphasic response was observed for fast oscillations (30–90 Hz) (Figs. 4B, S3 and S4). Initially, these oscillations decreased (up to −40 %), until the last hour of exposure when they increased (+ 25 %),
<table><caption>**Table 3**
Behavioral profile of rats following exposure to air (D4) and industrial solvents (D4'). All results are expressed as mean ± SD. *< 0.05; **< 0.001. The number of animals used for statistical analysis is indicated for each test conducted. Non-parametric tests on paired data (Wilcoxon Test, W) were performed following exposure to air (D4) and industrial solvents of interest (D4').**</caption><thead><tr><th rowspan="2">Test</th><th rowspan="2">Measured parameter</th><th colspan="3">TOLUENE</th><th colspan="3">STYRENE</th><th colspan="3">MEK</th></tr><tr><th>AIR (D4)</th><th>SOLVENT (D4')</th><th>n</th><th>W</th><th>p-value</th><th>AIR (D4)</th><th>SOLVENT (D4')</th><th>n</th><th>W</th><th>p-value</th></tr></thead><tbody><tr><td>Locomotor activity (Open Field)</td><td>Total distance [m]</td><td>19 ± 6</td><td>28 ± 7</td><td>12</td><td>66.0</td><td>0.006 **</td><td>19 ± 7</td><td>20 ± 4</td><td>12</td><td>30.0</td><td>0.266</td><td>18 ± 4</td><td>23 ± 11</td><td>10</td><td>13.0</td><td>0.556</td></tr><tr><td>Grip strength (Grip test)</td><td>Forelimb grip strength [gf]</td><td>779 ± 120</td><td>728 ± 78</td><td>13</td><td>-45.0</td><td>0.127</td><td>660 ± 147</td><td>682 ± 172</td><td>12</td><td>14.0</td><td>0.622</td><td>764 ± 122</td><td>755 ± 142</td><td>12</td><td>-3.0</td><td>0.951</td></tr><tr><td>Motor coordination (Locotronic)</td><td>Total crossing time [s]</td><td>3.2 ± 0.7</td><td>2.8 ± 0.8</td><td>13</td><td>-71.0</td><td>0.010 *</td><td>3.9 ± 0.8</td><td>3.9 ± 0.9</td><td>12</td><td>32.0</td><td>0.233</td><td>3.4 ± 0.9</td><td>3.4 ± 1.5</td><td>12</td><td>-2.0</td><td>0.969</td></tr><tr><td></td><td>Number of total errors [#]</td><td>4.5 ± 1.6</td><td>3.3 ± 1.6</td><td>13</td><td>-53.0</td><td>0.038 *</td><td>5.2 ± 2.0</td><td>4.1 ± 1.2</td><td>12</td><td>-46.0</td><td>0.072</td><td>5.6 ± 2.2</td><td>4.1 ± 2.7</td><td>12</td><td>-42.0</td><td>0.105</td></tr></tbody></table>

driven by low Gamma band activity (30–50 Hz). This activity remained elevated post-exposure.

Exposure to 600 ppm of styrene caused a decrease in the power of slow oscillations from the first minutes of exposure (down to about – 40 % for the Alpha band 8–12 Hz). This decrease persisted for about 3 h (Figs. 4B, S3 and S4). However, unlike at 1000 ppm, the power of Gamma oscillations did not decrease at the start of exposure, rather they increased by about 10 % compared to the control period, reaching approximately + 20 % after 6 h (Figs. 4A and 4B, S3 and S4). Overall, exposure to 600 ppm styrene caused a significant decrease in the total oscillation power during the first 2 h of exposure, followed by a gradual increase to reach approximately + 20 % after 6 h (Fig. 4C, Figs. 4D, S3 and S4).

Exposure to 200 ppm styrene resulted in power changes mainly during the first hour of exposure: a ~ 20 % decrease in the power of slow oscillations and a ~ 10 % increase of fast oscillations (Figs. 4, S3 and S4).

At 50 ppm (OEL for styrene), a similar ECoG profile as seen at 200 ppm was obtained, but with lower magnitude (Figs. 4, S3 and S4).

A supplementary exposure to 1000 ppm of styrene was conducted at the end of the protocol (on the same animals) to verify that the observed power changes were similar to those obtained at the beginning of the protocol. The same biphasic ECoG profile as before (Fig. 2 and Fig. 4) was observed during this exposure, with power changes of the same order of magnitude (data not shown).

**4. Discussion**

The aim of this study was to test ECoG as a potential means to monitor the effects of VOC exposure on CNS function and to determine its sensitivity relative to classical behavioral tests. Brain waves are highly conserved across mammals (Buzsaki et al., 2013), making them a reliable "translational" biomarker. Incidentally, ECoG changes are already used by pharmaceutical companies to assess the effectiveness and safety of CNS treatments (such as for Parkinson's disease) in animals (Dürmüller et al., 2001). As brain waves reflect postsynaptic neural activity, which depends on neurotransmission, the ECoG technique could be considered as a quantitative functional biomarker for real-time neurophysiological monitoring.

The results presented here demonstrate that styrene and toluene, two commonly used industrial VOCs, induce distinct alterations in brain activity in rats, and that ECoG, by allowing real-time monitoring of the temporal kinetics of the onset of effects and the corresponding recovery, is a suitable technique for the study of acute neurotoxic effects of VOCs on the CNS. The sensitivity of ECoG exceeds that of traditional sensorimotor tests.

Toluene, a key component in inks, glues, and paint thinners, poses significant workplace exposure risks. It is one of the most extensively studied industrial solvents, with proven behavioral and molecular effects (van Thriel and Boyes, 2022; Cruz and Bowen, 2021; ATSDR), 2017; INRS, 2021). To reduce health hazards, the French and EU Short Term Exposure Limit (STEL) is set at 100 ppm. In animals, toluene exhibits biphasic effects on locomotor activity, causing motor excitation at low concentrations and sedation or anesthesia at higher doses (Chan et al., 2004; Riegel and French. 1999; Himnan, 1984; Armenta-Reséndiz et al., 2019); it also provokes significant neurochemical disturbances (Bowen et al., 2006; Bale et al., 2005; Bale et al., 2002; Von Euler et al., 1987; Lopreato et al., 2003; Lo et al., 2009; Gerasimov et al., 2002). In addition, electrophysiological studies have shown dose-dependent changes in ECoG activity during exposure (Contreras et al., 1979; Takeuchi and Hisanaga. 1977; Ghosh et al., 1990; Naalsund, 1986).

In rats, the euphoric dissociative state induced by toluene results from inhibition of GABAergic neurotransmission, mimicking the action of ketamine (Bale et al., 2005; Beckley and Woodward, 2011). This inhibition triggers a marked increase in rapid oscillations (> 30 Hz) in ECoG recordings in both animals and humans (Lazarewicz et al., 2010;
<figure><img src="image_6.png" /><figcaption>**Fig. 4.** Power modifications (mean ± SD) of (A) slow oscillation power, (B) fast oscillation power, and (C) total oscillation power relative to the baseline period (first hour of air exposure) over time, for each day of exposure to air (in black) or to the styrene dose of interest (in color). (D) Mean global time-frequency analysis of ECoG signal recordings during solvent exposure relative to ECoG signal recordings during air exposure.</figcaption></figure>

de la Salle et al., 2016).

In agreement with these results, we found that exposure to 1000 ppm toluene increased Gamma oscillation power (30–90 Hz) and decreased slow-wave power (Delta to Alpha). A similar dual effect was previously reported by Takeuchi and Hisanaga (1977) but only at 2000 and 4000 ppm. Indeed, their recordings from the rat hippocampus during a 4 h exposure to toluene revealed increased beta activity at 2000 and 4000 ppm, and a reduction in theta frequency at 4000 ppm. At 1000 ppm, they reported no effect, possibly due to methodological limitations at the time (analog systems), which made it impossible to record fast oscillations, such as Gamma waves. Interestingly, the effects of toluene on human health suggest different effects depending on dose, with low-level exposure leading to depressant effects such as decreased performance, fatigue, and dizziness, whereas higher-level exposure can induce euphoria (Benignus, 1981). Our findings suggest we are in the toluene high-exposure phase, with mainly excitatory effects.

Styrene is known for its acute neurotoxic effects, including CNS depression (Costa and Manzo, 1998; Bond and Bolt, 1989; (ATSDR), 2010; INRS, 2019) and electrophysiological impact (Matikainen et al., 1993). It has also been reported to induce rapid neurochemical alterations related to the catecholaminergic pathway (Coccini et al., 1999). Given its extensive use in polymer and resin production, acute occupational exposure to styrene presents a significant health risk, which lead to the establishment of STEL of 46.6 ppm in France and 40 ppm in the U. S. Here, exposure to 1000 ppm of styrene caused a marked reduction in the power measured across all frequency bands (especially in the 2–12 Hz range) during the first 3 h, in agreement with the known depressive effect. Surprisingly, over the following 3 h, overall power gradually recovered, primarily driven by a strong increase in low Gamma power (30–50 Hz). During the final hour of 1000 ppm styrene exposure, the pattern of the power change was quite similar to that of the last hour of 1000 ppm toluene exposure. This biphasic ECoG profile was consistent across experimental phases #1 and #2, confirming its reliability. Since styrene oxide, a major styrene metabolite, affects the catecholaminergic pathway in a similar way to styrene (Bergamaschi

et al., 1997; Coccini et al., 1999), one may postulate that the observed biphasic ECoG profile might reflect an adaptive mechanism to monoaminergic system disruptions caused by styrene and its metabolites.

Although differing in amplitude and timing, exposure to 600 ppm, 200 ppm, and 50 ppm styrene produced similar ECoG profiles, all of which differed from the ECoG profile at 1000 ppm. Indeed, whereas 1000 ppm strongly depressed both fast and slow oscillations, lower concentrations (600 ppm, 200 ppm, and 50 ppm) increased fast oscillation power while reducing slow-wave power. The magnitude and duration of these two opposing effects diminished as concentrations decreased. Interestingly, the ECoG pattern at 600 ppm was reminiscent of that of toluene at 1000 ppm. It thus appears that exposure to styrene below 600 ppm may affect GABAergic neurotransmission in a similar manner to toluene at 1000 ppm. However, a mechanistic explanation of the ECoG profile observed here for styrene is beyond the scope of our study and would be purely speculative.

The significant ECoG modifications observed at 50 ppm (the current French STEL), although limited to the first hour of exposure, raise concerns about the existing occupational limits. While no definitive safety threshold can be established from these results, it seems reasonable to conclude that efforts to regulate airborne styrene levels in workplaces should be reconsidered to better protect workers' health.

MEK is one of the ten most widely used industrial organic solvents. Studies on acute MEK exposure have consistently failed to show significant CNS depressant effects in humans or animals (Kawai et al., 2003; Dick et al., 1992, 1989; INRS, 2020). As moderate CNS depression occurs only at very high concentrations, MEK is considered to be of low toxicity, which explains why the French and EU STEL is set at the relatively high concentration of 300 ppm. Here, exposure to 1000 ppm MEK had no effect on brain oscillation power. To our knowledge, there are no published studies reporting the effects of MEK on brain activity; we therefore have no reference point for comparison of our results. However, we can state that the lack of observed effects is consistent with the "limited" neurotoxic potential of this substance (INRS, 2020; Dick et al., 1992, 1989; Kawai et al., 2003).
The toxicity of industrial solvents on the CNS was also evaluated here by studying the vestibular reflex by measuring post-rotatory nystagmus (NPR). Our results showed that acute exposure to 1000 ppm of either toluene or styrene affected the networks involved in the nystagmus phenomenon by increasing the number and duration of saccades. Our findings are consistent with the work of Larsby et al. (1986) and Niklasson et al. (1993), who also reported that exposure to toluene and styrene led to an increase in nystagmus. They suggested that these results were due to modifications to cerebellar activity, particularly effects on the vestibulo-cerebellar circuit linked to altered GABAergic transmission, which is closely linked to the functioning of Purkinje cells. In support of this suggestion, some effects of toluene on the vestibulo-ocular system are blocked by the GABA antagonist gamma amino-valeric acid, which is a GABA antagonist (Niklasson et al., 1993).

Our study results indicate that PRN measurements were significantly altered in the same conditions as ECoG signals. This method therefore appears to be as sensitive and specific as the ECoG method. However, since the reflexes measured by PRN are primarily governed by excitation/inhibition loops involving glutamate and GABA, for substances acting on other types of neurotransmitters (such as styrene, which affects the catecholaminergic system), PRN may not be as relevant as ECoG.

Animals were subjected to behavioral tests for locomotion, sensorimotor coordination, and muscle strength (open-field®, Locotronic® and grip strength®, respectively) to compare the sensitivity of these tests with the ECoG results.

Locomotion alterations were only observed in rats exposed to 1000 ppm of toluene, with effects manifesting as a hyperactive behavioral profile. These observations are compatible with the ECoG effects reported, and concur with results presented by Arito et al. (1985), who observed hyperactivity following repeated toluene injections. Exposure to styrene and MEK had no significant effect on the animals' motor performance. This result was not surprising as the literature suggests that styrene affects the CNS and induces muscle weakness, drowsiness, incoordination, and tremors following exposure to at least 2500 ppm for 10 h (INRS, 2019), and that MEK has limited behavioral effects at this concentration. Nevertheless, we cannot rule out that behavioral tests involving more cognitive functions (such as social interactions, special learning, passive avoidance) would have provided more sensitive results. Indeed, Armenta-Reséndiz et al. (2019) reported that exposure to toluene for 30 min led to significant anxiolytic-like phenomena, learning issues and alterations to social interactions at concentrations of 2000 ppm and above. In our experimental conditions, it was both impractical and impossible to apply these complex tests immediately after exposure.

The behavioral results reported here, and especially the absence of clearly determined profiles (as in the case of ECoG), also highlight the potential experimental bias that behavioral tests might induce depending on the test chosen. In addition, the parameters surrounding the timing of exposure and performance of the behavioral test may have a significant effect. For instance, we might have seen a marked behavioral profile following 3 h exposure to styrene (when overall oscillations were decreased), rather than the slight to no behavioral changes after exposure for 6 h.

In this regard, the strength of the ECoG method is linked to the real-time readouts, revealing acute effects of volatile toxicants on the CNS under standardized conditions. This technique thus has the potential to provide higher sensitivity than traditional sensorimotor tests.

## 5. Conclusion

Our findings indicate that ECoG offers a precise, objective method to measure brain function during (and not after) acute exposure to toxicants. It is thus more sensitive than traditional sensorimotor tests. ECoG is a robust tool to track the kinetics of solvent effects on brain activity in single-day exposures. Moving forward, we plan to apply this method to

evaluate a wide range of industrial VOCs with known or suspected neurotoxic effects, to help identify the most hazardous substances in the workplace.

## Author contributions

Estefania Bernal Meléndez, Benoît Pouyatos, Frédéric Cosnier, Ludivine Wathier designed the study.

Emmy Joubert, Marlène Mascherin, Ludivine Wathier, Stéphane Grossman, Stéphane Viton and Frédéric Cosnier managed inhalation exposure and ensured precise monitoring of exposure conditions.

Estefania Bernal Meléndez, Stéphane Boucard, Laura Guenot, Lise Merlen, Benoît Pouyatos, Aurélie Thomas and Thomas Venet performed ECoG, PRN and behavioral experiments, and collected tissue specimens.

Marlène Mascherin assayed solvent levels in blood and brain. Estefania Bernal Meléndez and Thomas Venet performed the statistical analyses.

Estefania Bernal Meléndez and Benoît Pouyatos wrote the manuscript, the final version of which was critically read and approved by all authors.

## CRediT authorship contribution statement

**Guenot Laura:** Methodology, Investigation. **Boucard Stéphane:** Methodology, Investigation. **Grossmann Stéphane:** Methodology, Investigation. **Merlen Lise:** Methodology, Investigation. **Mascherin Marlène:** Methodology, Investigation. **Joubert Emmy:** Methodology, Investigation. **Wathier Ludivine:** Writing – review & editing, Methodology, Investigation, Conceptualization. **Viton Stéphane:** Methodology, Investigation. **Pouyatos Benoît:** Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Investigation, Formal analysis, Conceptualization. **Venet Thomas:** Writing – review & editing, Methodology, Investigation, Formal analysis. **Cosnier Frédéric:** Writing – review & editing, Methodology, Investigation, Conceptualization. **Bernal Estefania:** Writing – review & editing, Writing – original draft, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Conceptualization. **Thomas Aurélie:** Methodology, Investigation.

## Ethics approval

Animals were treated according to the ethical standards defined by the French Research and Safety Institute for the Prevention of Occupational Accidents and Diseases (INRS; facility accreditation: E-54-547-10) for animal health and care in strict compliance with European Union (EU) directive 2010/63/EU relating to the protection of animals used for scientific purposes. The regional ethics committee (Comité d'Ethique Lorrain en Matière d'Expérimentation Animale, CE n°66) approved the study protocol, which was subsequently authorized by the French Ministry of Higher Education and Research (Authorization number: APAFIS#21585).

## Consent to participate

Not applicable.

## Consent for publication

Not applicable.

## Formatting and funding sources

This work was entirely supported by the French Research and Safety Institute for the Prevention of Occupational Accidents and Diseases (INRS, France).
## Conflict of interest/Competing interests

The authors declare that they have no conflict of interest.

## Availability of data and material

All data generated or analyzed during this study are included in this article (and its Supplementary information files). The datasets analyzed are available from the corresponding authors upon request.

## Code availability

Not applicable.

## Declaration of Competing Interest

No conflict of interest.

## Acknowledgments

The authors gratefully acknowledge help with rat handling and husbandry from Armel Nijman, Aline Dosen and Laurine Douteau at the laboratory's animal facility.

The authors thank Dr. Maighread Gallagher for reviewing and correction of the paper.

## Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.neuro.2025.07.001.

## Data availability

Data will be made available on request.

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