大小鼠全身雾化暴露装置，动物全身暴露系统

1. Introduction
The antidegradant N-(1,3-Dimethylbutyl)-N′-phenyl-p-phenylenedi-
amine (6PPD) is a phenylenediamine compound widely used as tire and
rubber antioxidant to protect from oxygen and ozone (O3)[1,2]. Over
the last half century, the usage of tires has dramatically risen as the
number of automobiles worldwide has increased year by year. The mass
percentage of 6PPD in tires is about 0.4 %-2 %, and 6PPD persistently
enters the environment due to large amount emissions of tire wear
particles[3]. Currently, the global environmental emissions of tire wear
particles reached up to 5.9 million tons per year, and those tire wear
particles account for 3–7 % of the particulate matter (PM2.5) in the air,
increasing the risk of inhalation of 6PPD[4]. More seriously, environ-
mental factors such as ground-level O3, ultraviolet A (UVA), high tem-
perature, and high humidity can promote the aging processes of 6PPD[2,
5,6], which may produce toxic derivatives and further aggravate envi-
ronmental pollution and health burden.
6PPD and its oxidized products were first detected in water, with
environmental concentrations ranging from ng/L to μg/L levels, and
have attracted attention due to the ecological risk[7]. An increasing
number of studies have shown that environmental 6PPD and its aging
products can attach to dust particles, which can diffuse into the air to
form PM2.5 for long-distance transport[8,9]. The median total 6PPD
concentrations in road dust, parking lot dust, and vehicle dust collected
in southern Chinese cities were 226 ng/g, 232 ng/g, and 156 ng/g,
respectively, which were much higher than those in indoor dust
(14.0 ng/g)[10]. 6PPD-quinone (6PPDQ) is an important derivative of
6PPD after ozonation, and the total detection rate of the 6PPDQ in PM2.5
was 81 % in the urban environments of Taiyuan, Zhengzhou, and
Guangzhou, China[9]. 6PPD and 6PPDQ have also been detected in
PM2.5 in Hong Kong, China, and the daily intake of 6PPDQ for local
general population is predicted to be 1.08 ng/kg/day[11]. Moreover,
the detection rate of 6PPD and 6PPDQ in human urine in the South
China was as high as 60–100 %, and the concentrations of 6PPD and
6PPDQ in the urine of pregnant women (0.068–2.91 ng/mL) were
significantly higher than those in adults (0.018–0.40 ng/mL) and chil-
dren (0.015–0.076 ng/mL)[12,13]. In particular, workers in the rubber
industry and those in traffic-relevant occupations are at risk of exposure
to 6PPD and their quinone derivatives[14]. Considering the widespread
detection of 6PPD and its aging products in airborne particles and bio-
logical samples, long-term inhalation of those particles may pose a po-
tential health risk to human, and further investigations on the inhalation
toxicological effects on mammals are necessary.
The currently thoroughly studied aging product, 6PPDQ, has a me-
dian lethal concentration (LC50) of less than 1 μg/L for coho salmon[7],
and may be neurotoxic and cardiotoxic to zebrafish at the concentra-
tions of 10 µg/L[15,16]. In previous toxicity experiments, it has been
found that 6PPDQ can cause multiple organ injury in mice by gavage
(100 μg/kg) or intraperitoneal injection (0.4 mg/kg) for 28 days
[17–19]. Nevertheless, it should be notable that the main exposure route
for humans is inhalation of airborne dust containing 6PPD and its aging
products[9,20,21], while whose respiratory toxicity largely remains to
be investigated. The aging of 6PPD in environment is much more com-
plex, which generate products including phenol, 4-[(1,3-dimethylbutyl)
amino]-(4-DBAP), and 4-hydroxydiphenylamine(4-HDPA) more than
just 6PPDQ[22]. In addition to O3, ultraviolet light has also been re-
ported to accelerate the aging process of environmental contaminants
through photoaging and even affect their toxicity[5,6]. Currently, few
studies have examined the effects of O3 and UVA on the aging and
toxicity of 6PPD, further study is urgently needed to support the health
risk assessment and environmental management.
In this study, we constructed an artificial accelerated aging system to
investigate the effect of O3 and UVA on the aging process of 6PPD, and
C57BL/6 male mice were exposed to 6PPD and differently aging 6PPDs
to investigate the repeated inhalation toxicity heterogeneity at human-
comparable levels, including respiratory system, neurobehavior alter-
ation, and genetic damage. The study may contribute to the toxicolog-
ical knowledge of 6PPD and its aging products, and provides a novel
perspectives for the interference of environmental factors on the toxicity
of pollutants.
2. Methods and materials
2.1. Chemicals
6PPD (purity > 97.5 %) was purchased from Macklin (Shanghai,
China). 6PPDQ (purity = 97.8 %) was purchased from Dr. Ehrenstorfer
GmbH (German). 13C6-6PPDQ (purity > 98 %) was purchased from
Cambridge Isotope Laboratories (Massachusetts, USA). HPLC-grade
ammonium acetate, formic acid, and methanol were purchased from
Sigma-Aldrich (USA).
2.2. 6PPD aging reaction
Oxidative aging of 6PPD by O3 was conducted in accordance to
previous study[6,23]. Briefly, a total of 500 mg of 6PPD particles matter
were ground into dark gray fine powder. The 6PPD powder was then
spread on the bottom of a 1 L glass suction bottle and connected to the
O3 reaction chamber. A combination of oxygen generator and O3
generator (Guangzhou Chuavg Ozone Electric Equipment Co., China)
were used to generate O3. According to our previous methodology
(Table S1), the O3 flow rate was set at 0.5 L/min, in the oxidation aging
reaction of 6PPD, O3 concentration of 20 ppm was used to react with
500 mg 6PPD for 30 min for ozonation, and O3 concentration of
200 ppm was used to react with 500 mg 6PPD for 120 min for perozo-
nation. In UVA photoaging, the 6PPD powder was firstly spread on an
8 * 8 cm2 flat plate and placed in a chamber with UVA irradiation (in-
tensity of 300 μW/cm2) for 30 min, with a total irradiation dose of
34.56 J. After UVA photoaging, the 6PPD powder was further to be
oxidized by O3 aforesaid. After aging reaction, differently aging 6PPDs
was dissolved in 50 mL of ethanol and stored at −20 ℃.
2.3. 6PPDQ measurement and full wavelength scanning
6PPDQ concentration is determined to evaluate the ozonation degree
of 6PPD. According to previous studies[5,23], the concentrations of
6PPDQ in the ethanol stock resolutions were determined by using a
triple-quadrupole UPLC-MS/MS system (Acquity UPLC-Xero TQS,
X. Li et al.
Journal of Hazardous Materials 486 (2025) 137000
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