Review of Health Effects of Automotive Brake and Tyre Wear Particles

Abstract

Non-exhaust emissions from brakes and tyres are becoming the major transport-related contributor of particulate matter (PM) pollution in cities. Furthermore, tyre microplastics are the major contributor of unintentionally released microplastics in all environmental compartments. The European Union introduced for the first time worldwide limits for brakes (PM₁₀) and tyres (total abrasion mass) with the Euro 7 regulatory step. Thus, the interest in brake and tyre particles regarding health and environmental impacts has significantly increased in recent years. In this review, we summarise studies that assessed the impact of brake and tyre particles on human, mammalian, aquatic, and terrestrial cells and organisms. Furthermore, we summarise the studies that compared the impact of brake and tyre particles to other sources. We also critically examine the sampling methodologies of brake and tyre particles for health and environmental impact studies.

1. Introduction

Air pollution emissions have declined in the last two decades as a result of regulatory action in different sectors. For example, between 2005 and 2022, the number of premature deaths in the European Union (EU) attributable to particulate matter (PM) fell by 45% [1]. Premature deaths are projected to further decrease by 65% by 2030. Despite this improvement, air pollution remains the largest environmental health risk in Europe. Exposure to PM above the World Health Organization (WHO) recommendations caused an estimated 239,000 premature deaths in 2022 in the EU. While poor air quality is a critical issue that affects the whole world, it impacts some places more than others. For instance, 70% of all premature deaths caused by poor air quality happen in Pacific Asia [2]. Besides health issues, air pollution increases healthcare costs, reduces life expectancy, and damages vegetation and ecosystems, water and soil quality, and local ecosystems.

Pollutants of increasing concern are microplastics (synthetic polymer particles below five millimetres that are organic and insoluble and resist degradation) [3,4]. Microplastics are often added to products such as the granular infill material used in artificial sport surfaces, cosmetics, medicines, and detergents; they are also used in packaging. Tyre wear contributes significantly to the unintentional release of microplastics into the environment [5]. The smaller ones are emitted into the atmosphere, while the majority (coarse size and larger) are deposited on the road or nearby areas [6,7,8]. Resuspension, wind, and rain remobilise them. The majority of the particles settle in the soil; however, some end up in water via drainage systems (with or without treatment). Environmental aging processes such as thermooxidation, photooxidation, ozonolysis, shear stress, biodegradation, and leaching then take place, altering the physical and chemical properties of microplastics [9]. Recent work has highlighted the atmosphere’s role in transporting microplastics to remote locations [10].

Figure 1 plots the estimated European Union 27 (EU27) emissions of PM₁₀ and tyre microplastics into the environment for the years 2015, 2025, and 2035, assuming no mitigation actions [5,11]. Regarding PM, exhaust emissions will decrease significantly, leaving non-exhaust emissions as practically the main source of PM pollution. Electric vehicles still produce tyre particles, but they are expected to emit much fewer brake particles due to regenerative braking. However, it is always important to keep in mind that they do still produce brake wear particles, and corrosion over time that occurs as a result of fewer braking events might increase them. Brake particles have size distributions that peak at around 2–5 μm, which practically means most of the mass lies below 10 μm [11,12]. Tyre abrasion, which is in the order of 1000 kt per year, is the main source of non-exhaust microplastics [5]. In the EU, with Euro 7, worldwide non-exhaust emission limits were introduced for the first time. For brakes, PM₁₀ will be regulated, while for tyres, the total abrasion will be regulated.

The contribution of non-exhaust sources to the total PM depends on the contribution of other sources as well, such as power generation plants, industry, the sea, and biomass burning [13,14], but also on road maintenance, weather conditions, and driving conditions. Resuspended dust, which includes previously emitted non-exhaust particles, can have a big impact on the absolute PM levels. As an example, PM₁₀ levels can be significantly different in developed countries compared to countries where less attention is paid to the infrastructure [15]. While the exact composition of the tyres is an industry secret, they can be categorised into a few different groups. Summer tyres tend to be harder in nature in order to resist higher temperatures, while winter tyres tend to be softer in order to stay flexible in lower temperatures, and they usually have a larger, more complex tread pattern to support safe driving in wet conditions. Another commonly used tyre is the “all-season” tyre, which combines the technologies used in both summer and winter tyres in order to provide safe driving in all weather conditions. However, “all-season” tyres have caused some controversy, as it is believed that they provide more of a compromise between summer and winter tyres, not really being ideal for either season. Lastly, there are studded winter tyres which are used in many Nordic countries where the weather conditions are extreme. The hard metal studs installed in the tyre tread provide higher friction on slippery surfaces like icy roads. The general composition of tyre treads consists of rubber polymers (40–60%), reinforcing/filler agents (20–45%), and chemical additives (5–15%) [5]. Inorganic compounds such as Si, S, Zn, Ca, Al, and Fe and organic compounds such as benzothiazoles, N-(1,3-dimethylbutyl)-N′-phenyl-1,4-phenylenediamine (6PPD), and polycyclic aromatic hydrocarbons (PAHs) can be found in high concentrations. It is important to note that there is also a significant difference between light-duty and heavy-duty tyres and their composition. The particles found in the environment are tread particles encrusted and mixed with foreign materials from the road, dust, brakes, and soil.

The aim of a mechanical car brake system is to slow down or stop the vehicle by dissipating its kinetic energy and transforming it into frictional heat. In modern light-duty vehicles, two different kinds of brake systems are typically used: disc brake and drum brake systems. Drum brake systems consist of a spread mechanism that pushes two brake shoes against the inner surface of a cast iron or steel brake drum, connected to the wheel. A disc brake system consists of two main parts: the rotor and the pads. To allow a braking action to take place, the pads are pressed to the rotors. Because of their superior heat capacity, discs brakes are arguably safer than drum brakes on the front wheels.

The pad material differs depending on the region and its requirements. Non-asbestos organic (NAO) pads are commonly used in the US and Japanese market for comfort reasons. On the other hand, due to performance requirements, low-steel pads are common in Europe, together with semi-metallic ones, which have improved performance at high temperatures. This affects the chemical composition of the pad materials and potentially their toxic effects. To comply with the new Euro 7 regulations, hybrid pad materials that are a mix of NAO and low-steel pad ingredients have also been developed. Brake pads generally comprise five main components: binders (mainly phenol–formaldehyde resins), reinforcing fibres (copper, steel, brass, potassium titanate, glass, organic material (aramid), and Kevlar), fillers (barium and antimony sulphate, magnesium oxides and chromium oxides, silicates, ground slag, stone, and metal powders), frictional additives or lubricants, and abrasives (aluminium oxide, iron oxides, quartz, and zircon) [16,17,18]. Brake particles do not only originate from the pads but also from the disc which the brake pads are forced against. Discs used in passenger cars are typically made of grey cast iron, but in some cases, they can be made of reinforced carbon–carbon, ceramic matrix composites, or aluminium or even coated with materials that alter their properties and reduce wear.

Heavy-duty vehicles also use disc and drum brakes, in addition to different kinds of auxiliary brakes. Auxiliary brakes can be separated into engine brakes, electromagnetic auxiliary brakes, hydraulic auxiliary brakes, and compression release engine brakes. For the heavy-duty sector, the brake pad material composition differs for different market segments. For example, the bus segment prefers comfort and low noise emission, while long-distance trucks demand higher performance, i.e., higher coefficient of friction. New solutions are anticipated for the forthcoming Euro 7 regulation for the heavy-duty sector.

Many compounds from tyres and brakes have been found to be harmful, such as Zn, Cu, and PAHs [19,20,21,22]. Some tyre substances become more toxic when exposed to ultraviolet (UV) radiation. An example of such an oxidation product is 6PPD quinone [23]. There are a few reviews assessing the health and environmental impact of brake and tyre particles (e.g., [22,24] (and more will be presented in the next section)). However, a comprehensive review of the impact of both tyre and brake particles on mammalian, aquatic, and terrestrial organisms is missing.

Based on this background, the aim of this paper is to summarise the dedicated studies that have examined the impact of brake and tyre particles on health and the environment. Although there are reviews on this topic, studies have increased significantly in the last years. Furthermore, we try to understand whether non-exhaust particles are more or less harmful than other particles. Finally, we try to critically review the sampling methodologies in order to give recommendations for future studies.

2. Materials and Methods

2.1. Search Methodology

We searched articles in the PubMed and Google Scholar databases (accessed on 20 December 2024) using various keyword combinations, including “brakes”, “tyres”, “tires”, “non-exhaust”, ”particles”, “particulate matter”, “emissions” “health”, “human”, “aquatic”, and “terrestrial”.

Only original articles published in English and accessible through open access or institutional subscriptions were considered. The initial search yielded many articles, which were subsequently filtered by reading the titles and abstracts. In addition to research articles, we separately assessed reviews on the topic. Furthermore, relative papers cited in those studies were also checked.

Table 1. Reviews on health impacts of non-exhaust particles.

Year	Ref.	Brakes	Tyres	Comments
2018	[9]	N	Y	Aquatic environment
2020	[31]	N	Y	Human effects
2022	[32]	Y	N	Human effects
2023	[33]	Y	N	On mammalian models
2023	[24]	N	Y	In vivo and in vitro studies
2023	[34]	N	Y	Only urban parks
2024	[35]	N	Y	Environmental impact
2024	[22]	N	Y	Mostly ecological and ecotoxicological
2024	[36]	N	Y	Leachates
2024	[37]	N	Y	Aquatic and terrestrial effects
2024	[38]	N	Y	Environmental and health

summarises the key reviews on the topic. Reviews of microplastics are not included (e.g., [25,26,27,28,29,30]).

2.2. Methods of Toxicological Studies

In this paper, we looked at toxicological studies, as there were almost no epidemiological studies published on the health effects of non-exhaust emissions. The main reason for this lack of epidemiological studies is the ethical concerns around it. Long-term studies exposing humans to potentially harmful emissions, in order to study the effects, are not ethically correct as they would cause direct harm to the subjects involved. This is why most of the current studies are conducted on cells, mice, and other organisms. There are a few epidemiological studies looking at observational data; however, with such studies, it is often hard to establish a direct causal relationship between non-exhaust emissions and health outcomes, as humans are exposed to various different sources of pollution. This is why it is crucial to find certain markers that can point to these sources of pollution. Some studies indicated that some compounds typically found in brake particles (e.g., Cu) or tyres particles (e.g., Zn) are associated with adverse cardiovascular health outcomes or mortality [12,39,40,41]. On the other hand, one epidemiological study used barium as a marker for brake wear particles, which shows that markers do not necessarily prove the presence of a certain pollutant [42].

Another important observation is that a proportionally higher number of toxicological studies were conducted in vitro compared to in vivo. This happens because in vitro studies are much easier and cheaper to perform and have less limitations on exposure pathways and dosages. Studies on mammalian organisms were carried out using human or mice cells. On the other hand, most of the studies on aquatic or terrestrial organisms (not mammalian) were conducted in vivo.

Overall, most of the toxicological studies followed a similar methodology. For the in vitro studies, the exposure methods were either submersion or an air–liquid interface. On the other hand, in most in vivo studies, the exposure method was either inhalation or (intratracheal) installation. The benefit of intratracheal installation is that it directly deposits the substance in the airways, and it is much easier to control the exposure dosage. However, this method can often cause disturbances and trauma to the subject, which could, in turn, affect the results of the study [43,44,45].

The main parameters used to assess the toxicity of wear particles were the following:

• Reactive oxygen species (ROS), which are chemically reactive molecules containing oxygen often generated in response to environmental stressors.
• Interleukine-8 (IL-8), which is a cytokine (protein) produced by different cells in the body including immune cells and involved in the regulation of inflammation and the immune response.
• Tumour necrosis factor alpha (TNF-α), which is a cytokine (protein) produced by immune cells (macrophages, monocytes, etc.) and involved in the regulation of inflammation and the immune response.

In some of the in vivo studies assessed, the subjects’ behavioural changes were also noted, for example, the swimming frequency and pattern of fish. This information can be extremely insightful, by showing us how the compounds can affect the subjects’ nervous system, motor skills, and overall wellbeing. However, behavioural changes alone cannot give the full picture, and they should always be used in combination with toxicological tests. This is the case because behaviour can easily be affected by the stress and trauma caused to the subjects during the administration process; hence, there should always be precautions in place in order to minimise the stress caused. Some of these precautions include habituation, maintaining a constant environment, and proper handling. Nevertheless, in some cases, such as intratracheal installations, there is little that can be done in order to minimise the stress caused to the subject.

2.3. Methods for Brake Sample Collection

The main methods used to collect brake samples were as follows:

• Brake bench dynamometer.
• Box around brakes.
• Brake drum or brake lining of vehicles.
• Room filter of brake bench dynamometer installation.
• Grinding of brake pads.

It is crucial to take note of how the emission samples were collected in these studies in order to figure out if the results are realistic and representative of real life. Many of the brake wear particle samples were not collected using a representative methodology. In laboratory sampling, the conditions used were often extreme in order to collect the samples quickly; however, this means that the morphology and composition of the samples were not representative of real emissions. When the same brake pad goes through multiple braking cycles without appropriate pauses and ventilation, and the temperature and pressure are extremely high, physical and chemical reactions which normally do not take place can happen and possibly create new compounds. There are situations where these extreme conditions might occur; however, it is important to be realistic about the probability of the general population to be exposed to these types of particles, in order for them to cause harm. Most of these extreme driving conditions happen outside of urban environments, often in scarcely populated areas or on racing tracks. For example, wear cycles like AO4D and SAE J2522, which were designed to assess the effectiveness behaviour of a friction material, result in extreme driving conditions and high temperatures [46,47], which are unrealistic for passenger vehicles.

One of the most realistic sample collection methods, out of the ones mentioned in this review, was carried out in a bench dynamometer test chamber [48]. In this study, three different brake pads were tested (low-steel, high-steel, and NAO brake pads). All the brake pads were run through three different brake cycles: two realistic, or mild, conditions and one severe. The first cycle consisted of 8 braking events of mild deceleration, 120 to 80 km/h, and a temperature of 100 °C. The second cycle consisted of 8 braking events of more intense deceleration, 200 to 170 km/h, and a temperature of 100 °C. The last cycle consisted of 15 braking events of extreme deceleration, 100 to 5 km/h, and reached temperatures of 550 °C. The severe cycle reached extreme temperatures which would not normally occur during real-life driving conditions. However, it must be noted that the particles collected from all three cycles were then combined and the organic parts extracted. This means that, although two of the cycles run were realistic, the final sample contained a large proportion of particles from the extreme cycle and hence is not representative of the majority of real-life emissions. Furthermore, only the organic extracts were used for the toxicology assessment, so the results of this study cannot be considered representative of brake wear emissions as a whole.

In one 2009 paper [49], the particles used were freshly collected through an exposure box installed around one brake of a car. The cell lines used to assess the health effects of brake wear particles were placed directly inside the exposure box in order to simulate real-life exposure. This is a much more realistic way to assess the toxicity of fresh particles, which are usually the type of particle humans are exposed to in urban environments. However, in this specific study, the vehicle was installed on a garage lift, so the tyres were not touching the pavement, and the forces involved in the braking procedure were not necessarily realistic.

Another method used is collecting the sample from the brake drums of vehicles after prolonged use. For example, in one study, samples from the brake drums of seven passenger vehicles with an average age of 10 years and average distance of 51,732 km were collected [50]. Although this method is fairly realistic, it has two main flaws. Firstly, the samples collected are not necessarily representative of real emissions, as the brake drums are not completely closed and can allow for some of the smaller size fractions to escape, as well as let particles from other sources, like tyre and road wear or exhaust emissions, enter. Secondly, some of the particles collected were released a long time before sample collection, which means that these aged particles could potentially have a very different composition and morphology to the original emissions, due to processes like oxidation and agglomeration, skewing the results once again. Thus, the results of such studies on aged particles (e.g., [50]) may be different compared to other studies on fresh particles, probably due to the highly oxidised and aged state of the particles and their larger size. Nevertheless, research comparing aged and fresh brake particles is of interest. The researchers also collected samples from the brake linings of three different vehicles after use; however, not much more information is given about the sample collection.

Another study collected samples from the filter of a brake testing laboratory, containing a year’s worth of particles [51]. Like before, the samples used in this study might not be representative of real-life emissions, as the particles were aged and also because they came from a laboratory, through a variety of testing cycles and possibly unrealistic driving conditions.

Probably the least representative sample collection method is the grinding of brake materials. The particles released during real braking scenarios are different from the particles created by grinding the same materials. During typical braking behaviours, the temperatures rise significantly, and chemical reactions can occur, creating different compounds. This is shown in a series of studies [46,52] in which particles collected from a road simulator and ball-milled particles were both assessed. In both studies, the ball-milled samples caused a mutagenic response, whereas the friction samples did not cause any significant response, which further highlights the difference in the particles produced by different methodologies.

Out of the papers mentioned in this review, only four [52,53,54,55] carried out in vivo experiments, unfortunately using unrealistic sample collection, highlighting the need for more research on the topic. To this date, there has not been an in vivo study using a representative sample collection methodology published yet, in order for the results to be able to be taken into consideration when assessing the potential toxicity of brake wear particles. This means that although the results produced from these papers are important to take into account, they cannot be used to determine the real adverse effects on human health.

2.4. Methods for Tyre Sample Collection

The main methods used to collect tyre samples were as follows:

• Laboratory road simulator.
• Cryo-milling or cryo-fracturing of tyres.
• Grinding, shredding, or scraping of tyres.
• Pre-made crumb rubber (with unknown grinding technique).

The main challenge when it comes to collecting tyre emissions is that, unlike drum brakes, for example, the particles are not collected anywhere, and they are released directly into the environment, which makes it especially hard to collect them during on-road testing, which would be the most realistic. Laboratory testing with a road simulator consists of tyres mounted on a rotating axis and a round pavement for the tyres to run on. Although this method is convenient, it is not completely accurate, as the tyres are constantly at a slight angle, and there are no perpendicular forces applied like there are in real-life driving. This does not represent real-life driving conditions where the tyres are constantly changing direction. The representativeness of the particles emitted from tyres in a road simulator is also dependent on the conditions used during the testing and the pavement composition, although, unlike brakes, the temperatures reached are not extremely high, and so there should not be a significant difference.

Many studies rely on other methods to collect particles that are much faster and cheaper but, unfortunately, much less realistic. One of the methods used the most is cryo-milling or cryo-fracturing. This method consists of taking scraps or small parts of tyres and freezing them with liquid nitrogen in order to make them brittle, without altering their composition, and then ball milling them until they reach the desired particle size. Other similar methods are grinding, shredding, or scraping the tyre to create particles. Although these processes are very convenient, as they allow for a lot of control, they are not at all representative of the real-life process of tyre abrasion and the particles emitted into the environment. One more method used in some of the papers is rotating a tyre against a steel brush [56]. This method is also not completely accurate; however, it is slightly more realistic than grinding and ball milling, as the particles released are created by abrasion.

In all but three of the studies carried out on aquatic organisms, the samples used were not representative of real emissions, as most of the samples were obtained from cryo-milling or cryo-fracturing, shredding and grinding, or crumb rubber materials. And out of the studies carried out on terrestrial organisms, none of them were carried out using realistic sample collection methods. On the other hand, in the studies conducted in mammalians, eight of them used samples collected in a road simulator. In the studies carried out on aquatic organisms, the researchers seemed to value more the time and cost efficiency that techniques like cryo-milling provided, whereas in the studies carried out on mammalian organisms, the researchers seemed to want to obtain the most realistic results, in order to have an accurate representation of the health effects on humans.

In the aquatic studies, the only ones that used realistic sample collection were conducted by collecting samples from a road simulator laboratory in an interior drum testing system with asphalt pavement in cassettes [57]. Considering the fact that their studies are the only ones carried out with representative samples, it is interesting to see that the results only showed mild toxicity and no significant effects on the growth or reproduction of non-salmonid species.

In many of the studies conducted on aquatic organisms, the exposure was conducted through leachates, sediments, and elutriates. This is mostly performed because the exposure mechanism for aquatic organisms is usually through particles entering the water system. Many different leaching methodologies were used. One study even used sequential leaching to see how that would change the toxicity.

3. Results

Figure 2 summarises the studies and their conclusions regarding toxicity. The papers were separated into the ones that showed no adverse health effects, the ones that presented few health effects or not extremely harmful ones, and the ones that showed significant or detrimental adverse effects on the organisms studied.

3.1. Brakes

Table A1. (a) Studies investigating the impact of brake particles on human/mammalian tissues. (b) Studies investigating the impact of brake particles on aquatic species.

(a)
Year	Ref.	Source	Parameters Examined		Testing Conditions	Main Conclusions
2009	[52]	Prototype SM brake pad material emulating LM European brake system.	Brake wear debris (non-airborne) and ball-milled dust in vitro mutagenic potency and in vivo pulmonary toxicity via different types of assays.		Laboratory brake wear sampling (non-airborne dust) on a brake dyno in A04D wear test. Brake temperature up to 500 °C.	Brake wear debris proved toxic to E. coli strain after metabolic activation (Cu). No other significant biological effects were observed. Not clear if the prototype pads are representative of real-world commercial brakes.
2009	[49]	Brake system of one vehicle—does not specify what kind of vehicle or the brake type.	Brake wear particles and debris in vitro toxicological effects on epithelial lung cells.		Enclosed chamber with direct exposure of the cells to freshly generated brake dust. Two braking protocols were applied.	Increase in oxidative stress and pro-inflammatory response in lung cells was observed with fresh brake particles. No cytotoxicity or cell morphology deterioration.
2015	[50]	Different drum brakes from passenger cars. Mixed brake dust samples of light-duty brakes.	Brake wear debris and aged dust ability to generate reactive oxygen species (ROS).		Deposited dust on the drum brakes of various light-duty vehicles. Representative of urban driving and high-speed braking.	Aged brake wear particles are not extremely potent in forming ROS.
2015	[53]	Brake dust alone and in combination with added chrysotile and crocidolite asbestos.	Fate and pathological response in the lung and pleura of brake dust following short-term inhalation exposure in vivo.		Grinding of chrysotile-containing brake drums.	The crocidolite asbestos fibres were persistent through the lifetime and produced an inflammatory response in the lung, whereas the brake dust alone and brake dust with chrysotile samples were biosoluble and caused no significant pathological response.
2016	[47]	Prototype SM brake pad material emulating LM European brake system.	Brake airborne PM in vitro genotoxicity assay in human peripheral lymphocyte cells.		Laboratory brake wear sampling on a brake dyno with AK-Master. Brake temperatures up to 550 °C result in high particle number.	Exposure to brake particles has an impact on DNA damage of lymphocytes. Presence of crystalline metal is considered relevant. Not clear if the prototype pads are representative of real-world commercial brakes.
2016	[46]	Prototype SM brake pad material emulating LM European brake system—two commercial brake systems were also ball-milled.	Brake wear debris (non-airborne) in vitro toxicity and mutagenicity via different types of assays.		Laboratory brake wear sampling (non-airborne dust) on a brake dyno with A04D wear test. Brake temperature up to 500 °C.	The non-airborne debris of the model material contained compounds with toxic and mutagenic properties. Not clear if the prototype pads are representative of real-world commercial brakes.
2018	[54]	Chrysotile-containing brake dust and chrysotile or crocidolite asbestos.	Dose–response and fate in the lung and pleura of brake dust in a 28-day in vivo quantitative inhalation toxicology study.		Grinding of brake shoes.	Chrysotile samples caused a slight inflammatory response, but the fibres were able to be cleared by alveolar macrophage clearance. The crocidolite fibres were not able to be cleared, and they caused a significant inflammatory response and mesothelial pathology. The brake dust samples showed no significant pathological response.
2018	[87]	Different types of brake systems (different pads) used in passenger cars.	Brake PM and dust in vitro toxicity in human epithelial and primary immune cells.		Laboratory brake dust sampling on a brake dyno under urban driving conditions. High brake temperatures were observed up to 500 °C.	Airborne LM brakes do not induce any adverse biological effects in the in vitro lung multicellular model. Bigger particles from NAO brakes caused inflammatory responses and affected cell viability and morphology. Presence of TiO2 is considered relevant.
2018	[88]	Different types of brakes mounted on light-duty vehicles and tested on a brake dynamometer.	In vitro toxicity of brake wear debris, aged dust, and nanoparticles in the respiratory epithelium.		Deposited dust on the wheel parts of light-duty vehicles and a brake dynamometer.	Short-term loss of viability but with limited pro-inflammatory effects. Cytotoxicity of brake wear seems to be particle-size-independent. Substantial amount of nanoparticles with metallic content is considered relevant.
2019	[80]	Different brake systems (LM, SM, NAO, hybrid pads) and studded tyres.	Brake and tyre PM2.5 in vivo toxicity through exposure in mice.		Lab for both sources. PM2.5 sampling on a brake dyno. Unrealistic highway testing sequence—tyre wear particles on a road simulator under normal driving conditions (speed of 70 km/h).	No significant responses observed. No cytotoxicity or oxidative stress was observed. Different potency to induce inflammatory responses by the different types of brakes.
2020	[51]	Mixed brake dust samples from different types of drum brakes of heavy-duty vehicles.	Brake wear debris and aged dust in vitro toxicity in human airways through exposure to airway macrophages.		Deposited dust on the drum brakes of various heavy-duty vehicles. Representative of urban driving and high-speed braking.	Similar toxicological profiles of brake and diesel samples. Both trigger cytokine secretion, impair phagocytic capacity, and disrupt mitochondrial integrity. The metal content of brake dust is considered relevant.
2020	[59]	Four different brake pad/disc systems of light-duty vehicles.	Brake wear PM2.5 in vitro biological impacts.		Brake dynamometer under realistic driving/braking conditions.	PM2.5 with higher Cu induced cell toxicity that correlated with Cu concentration.
2021	[55]	Chrysotile-containing brake dust compared to TiO2, chrysotile, crocidolite, or amosite asbestos.	Dose–response and fate in the lung and pleura in a 90-day in vivo quantitative inhalation toxicology study.		Grinding of brake shoes.	Brake dust and chrysotile showed no significant pathological or tumourigenic response; however, crocidolite and amosite showed inflammation, microgranulomas, persistent fibrosis, and a dose-related lung tumour response.
2023	[48]	Different types of brake systems (different pads including LM, SM, and NAO pads).	In vitro cytotoxicity of brake wear PM10-bound PAHs and plasticisers in human alveolar epithelium cells.		Laboratory brake wear sampling on a brake dyno with one severe and two milder braking protocols. Brake temperature up to 550 °C over the severe cycle.	Brake PM from low-steel, high-steel, and non-asbestos organic brake pads decreases cell viability. No increase in intracellular ROS and no cell cycle arrest were observed.
2023	[89]	Different types of brakes mounted on light-duty vehicles and tested on a brake dynamometer.	In vitro toxicity of brake wear debris, aged dust, and nanoparticles in the respiratory epithelium.		Deposited dust on the wheel parts of light-duty vehicles and a brake dynamometer.	Brake wear nanoparticles do not cause overt cytotoxicity and inflammation. They can translocate through the epithelial barrier and increase mucus production. Indication of acute inflammation and potential for chronic obstructive pulmonary diseases.
2023	[58]	Different types of brake systems of 3 vehicles.	In vitro toxicity of unspecified brake dust sample of up to 50 μm in a bronchial epithelial cell line.		Brush deposited dust from the brake discs of 3 vehicles. Very high Cd concentrations indicate use of old brake linings.	Brake dust demonstrates cytotoxicity and a significant secretion of the pro-inflammatory cytokine IL-8. Negative effects were attributed to Cu and Cd. No oxidative stress was noted.
(b)
Year	Ref.	Source	Species	Parameters Examined	Testing Conditions	Main Conclusions
2017	[81]	Brake system of three vehicles.	Echinogammarus Veneris (amphipod crustaceans).	In vivo assessment of the genotoxic and oxidative stress effects of brake wear debris on Echinogammarus veneris.	Collected from the brake linings of three different vehicles after use.	Brake dust was shown to cause less genotoxic damage than the natural dusts. Oxidative stress was demonstrated for brake dust.

of Appendix A summarises the studies that examined brake particles. More details about the studies can also be found in Appendix A. From the sixteen mammalian studies assessed, one showed no effects [46] (the ball-milled samples were mutagenic but not the friction samples), five showed significant effects, and ten showed some effect. Overall, the three studies that used realistic testing methodologies [49,50,58], i.e., sample collection from drum brakes after prolonged use, all showed some toxicological effects and pro-inflammatory responses, mostly ROS production and IL-8 secretion. It is important to note that most of the studies observed a concentration-dependent or dose-dependent response. For example, a study that exposed cells to fresh particles through an exposure box noted an increase in IL-8 levels with an increasing number of braking events and strength [49].

Another study suggested that the overall oxidative potential of brake wear particles can lower over time, since the ROS generation they observed studying aged brake wear particles (average 10 years) was lower compared to their previous study that tested fresh particles [50]. This is probably due to the highly oxidised state of these aged particles and because of their larger size, due to agglomeration, and smaller reactive surface area.

Many studies seem to suggest that the toxicity of brake wear particles is partly due to the presence of certain metals and heavy metals, known to be cytotoxic. This is further validated by the use of metal chelators, which bind to the metal ions forming a complex and removing the metals from the system. A study observed that after PM exposure, in the presence of a metal chelator, the initial heightened IL-8 and TNF-α secretion, as well as the impairment of phagocytic capacity, was completely abolished [51]. Furthermore, in a 2020 study, brake pads with different Cu concentrations were tested [59]. A correlation between Cu concentration and cell toxicity was found. However, it should be noted that it is extremely hard to prove that the toxicity is caused specifically by one component of the particles. For example, in one study, it was not possible to correlate the toxicological effects with the composition of the particles of low-steel, high-steel, and NAO brake pad samples [48].

When looking at the studies that did not use representative sample collection methods (e.g., [54]), most of them produced mixed results with no clear verdict on whether or not the particles produce significant toxicological responses. Overall, it is evident that brake wear particles are not intrinsically toxic; however, the size, composition, and exposure dose are what makes them dangerous to human health.

Some studies tested only asbestos-based brakes, which have been banned in Europe and the United States of America (USA) since the late 1990s due to health concerns [53]. Although not relevant to EU policy, there are, however, many countries that still use asbestos-based brake pads and linings to this day. Studies on asbestos-based brake materials confirmed what was already known about asbestos materials. Brake dust containing chrysotile showed no to little pathological response, as it is biosoluble and was able to be cleared from the lungs. On the other hand, crocidolite- and amosite-containing brake dust caused significant inflammation and a pathological response, as the fibres were not able to be cleared from the lungs. This happens because chrysotile particles are long and curly, whereas crocidolite and amosite fibres are known for their short and pointy shape, which makes them much more of a hazard, showing that the shape and size of a particle can make it more dangerous to human health.

3.2. Tyres

Table A2. (a) Studies investigating the impact of tyre particles on human/mammalian tissues. (b) Studies investigating the impact of tyre particles on aquatic species. (c) Studies investigating the impact of tyre particles on terrestrial species.

(a)
Year	Ref.	Source	Parameters Examined		Testing Conditions	Main Conclusions
2005	[90]	Tyre debris eluates and organic extracts.	Tyre debris toxicity in A549 human alveolar cells and HepG2 human liver cells in vitro and in X. laevis embryos in vivo.		Laboratory through cryo-fracturing of tyre scraps.	Dose-dependent increase in DNA damage and decreased cell proliferation. Morphological changes. Time-dependent increase in Zn in HepG2 cells. Increased mortality rate in X. laevis embryos and malformed larvae.
2005	[56]	Tyre debris organic extracts.	Tyre debris cytotoxicity in A549 human alveolar cells.		Laboratory by rotating a new tyre against a steel brush.	Dose- and time-dependent inhibitory effect on the reduction of MTT and dose-dependent increase in cell mortality and DNA strand breakage. Multiple structural alterations.
2006	[66]	Studded tyre wear PM10 across two different pavements compared to other sources.	Studded tyre wear particle inflammatory response in human macrophages and nasal and bronchial epithelial cells.		VTI road simulator using studded tyres and two types of pavements: dense asphalt with granite and stone mastic asphalt with quartzite.	Granite pavement caused a higher cytokine release compared to quartzite. Tyre particles caused a higher decrease in cell viability compared to street and subway particles. None of the samples caused a cytokine release from nasal epithelial cells.
2006	[82]	Tyre–road wear particles compared to other sources (studded and winter tyres).	Genotoxicity of studded tyre–road wear particles in A549 human alveolar cells.		VTI road simulator using 1 studded tyre on ABT pavement, 2 studded tyres on ABS pavement, and 1 friction (winter) tyre on sanded ABS pavement.	All particles were able to cause DNA damage; however, winter tyres on sanded ABS pavement caused significantly higher DNA damage. Studded tyres on ABT pavement caused an increase in IL-8 and TNF-α, and studded tyres on ABS pavement caused an increase in IL-6, IL-8, and TNF-α.
2007	[67]	Studded tyre wear PM10 across two different pavements compared to other sources.	Studded tyre wear particle inflammatory effects on mouse macrophage cells.		VTI road simulator using studded tyres and two types of pavements: dense asphalt with granite and stone mastic asphalt with quartzite.	All particles were able to induce cytokine release. Granite caused much higher release of TNF-α and IL-6 compared to quartzite. Both granite and quartzite induced similar lipid peroxidation and ROS formation, but only granite induced NO production.
2007	[63]	Tyre debris organic extracts.	Tyre debris cytotoxicity in A549 human alveolar cells.		Laboratory by rotating a new tyre against a steel brush.	Dose-dependent increase in % of Trypan Blue-positive cells. Time- and dose-dependent increase in LDH and lipid microdomains. No difference in cell proteins. Twenty-fold increase in HO-1 content in DRM. Increase in invaginations but no other morphological differences.
2008	[91]	Tyre debris organic extracts.	Tyre debris toxicity in A549 human alveolar cells (ROS production and Hsp 70 expression).		Laboratory through cryo-fracturing of a new tyre.	Significant dose-dependent ROS production. Higher Hsp70 expression with higher exposure times and lower doses.
2008	[68]	7 combinations of tyres, pavements, and traction materials compared to other sources.	Tyre wear particle toxicological effects on human monocytes.		VTI road simulator using studded tyres, non-studded winter tyres, ABT with granite, ABS with quartzite, no traction material, crushed stone as traction material, and natural sand as traction material.	All particles were able to induce a cytokine release. Granite caused much higher release of TNF-α, IL-8, and IL-6 compared to quartzite.
2008	[60]	Studded tyre–road wear particles compared to other sources.	Studded tyre–road wear particle genotoxicity through in vitro experiments on human lung epithelial cells.		VTI road simulator using studded tyres and ABT pavement.	Tyre–road samples caused the least amount of mitochondrial depolarisation (37%) and showed almost no increase in ROS.
2008	[43]	Tyre particles and metals (Zn and Cu).	Tyre particle cardiopulmonary toxicity in mice through intratracheal installations.		Laboratory by grinding fresh tyre material (recycled styrene butadiene rubber) and 2 tyre scrap samples.	There was a higher increase in total lavageable cells in the scrap sample. The Zn + Cu sample caused a much higher effect compared to the individual metals. All samples increased BALF lung injury markers and decreased mitochondrial aconitase activity.
2009	[44]	Tyre wear particles, PM2.5 and PM10.	(Size-fractioned) Tyre particle lung toxicity in mice (in vivo through intratracheal instillation).		Laboratory through cryo-fracturing of tyre scraps.	PM2.5 showed stronger cytotoxic effects, although PM10 did show some cytotoxicity at higher doses. PM2.5 was more dispersed in alveolar spaces and was not able to be cleared out.
2010	[45]	Tyre wear particles, PM2.5 and PM10, compared to other sources.	(Size-fractioned) Tyre particle lung toxicity in mice (in vivo through intratracheal instillation).		Laboratory through cryo-fracturing of tyre scraps.	TP2.5 reached the alveolar spaces and caused an acute inflammatory response, whereas TP10 reached bronchial spaces and did not cause a significant response.
2011	[92]	Studded tyre wear, PM10.	Tyre wear particles’ toxicology through protein release from human macrophages after TWP and ETX exposure.		VTI road simulator using studded tyres and dense asphalt pavement with granite.	TWP exposure: 7 down-regulated and 3 up-regulated proteins. Proteins involved in inflammatory response were up-regulated, and proteins involved in cellular functions were down-regulated. ETX exposure: 4 down-regulated and 2 up-regulated.
2012	[57]	Tyre and road wear particles.	Tyre wear particle lung toxicology through in vivo inhalation in rats.		Laboratory drum testing system with asphalt pavement using 1 summer silica tyre, 1 winter silica tyre, 1 carbon-black tyre.	No general toxicity, cytotoxicity, or inflammatory potential was observed. A few focal areas of subacute inflammatory cell infiltration were observed at higher doses.
2019	[93]	Truck and passenger tyre wear particles.	Tyre wear particles’ genotoxicity in mice macrophage RAW264.7 cells.		Laboratory through cryo-fracturing of a passenger tyre and a truck tyre.	Both samples caused a reduction in cell viability, decrease in metabolic activity, and increase in TNF-α. Truck tyres overall caused less toxicity than passenger tyres.
2020	[61]	Microplastics from polymer pellets and truck tyre materials.	Polymer and tyre microplastic ingestion toxicity in vitro through a 3D intestinal model.		Laboratory through cryo-fracturing of polymer pallets and tyre materials.	No significant cytotoxicity or release of pro-inflammatory cytokines was observed. There was no change in the barrier integrity of the co-cultures.
2022	[94]	Photoaged tyre wear particles (environmentally persistent free radicals).	Photoaged tyre wear particles’ toxicology through in vitro experiments on mice macrophages.		Laboratory through cryo-fracturing of tyre scraps.	TWPs caused a decrease in cell viability and an increase in ROS generation and mRNA levels for inflammatory factors. The toxicological effects were stronger with higher irradiation time suggesting that free radicals play a role.
2022	[62]	Road dust and tyre wear particles’ organic extract (PAHs).	Road dust mutagenicity with bacterial assays (Ames test).		VTI road simulator using 2 different summer tyres and with in situ resuspension chambers in 2 cities.	No mutagenic response was observed, although the testing conditions were not satisfactory, so the results are not reliable.
2022	[95]	Tyre wear microplastic particles.	Tyre wear particle lung toxicology through in vivo inhalation in mice.		Laboratory through cryo-fracturing of a tyre.	TWP exposure caused restricted ventilatory dysfunction and fibrotic pathological response in mice. Major histopathological changes were observed in the lungs consistent with pulmonary fibrosis. Cytoskeleton rearrangement and cell migration were observed in vitro.
2023	[69]	Tyre wear particles.	Tyre wear particle lung toxicity through a 3D in vitro model of human airway organoids.		Laboratory through cryo-fracturing of a tyre.	TWPs induced significant cytotoxicity and ROS generation and increased cytokines such as TNF-α and IL-6. Particles were found inside the cells and clustered around the cells. The exposed hAOs decreased in size. TWPs caused an increase in early and late apoptotic cells.
2023	[64]	Tyre wear particles.	Tyre wear particles’ toxicity in mice macrophage RAW264.7 cells.		Laboratory on-road simulator and then cryo-milled.	There was no sign of DNA damage, and toxicological responses were only observed after prolonged stress and high concentrations in the proliferation study, the gene expressions, and the immunoblot analysis.
2023	[70]	6PPD and 6PPDQ.	6PPD and 6PPDQ hepatotoxicity in mice in vivo.		-	Both substances were found to bioaccumulate in the liver in a dose-dependent manner. Higher doses increased the liver’s weight and the triglyceride levels. The hepatic metabolism and immune response were altered.
2023	[71]	6PPDQ.	6PPDQ toxicology in vivo in mice through intraperitoneal injections.		-	In the single-injected mice, there were no significant changes in organ indexes or biochemical parameters, and no pathological changes were observed. Repeated injections caused many changes in organ indexes. Significant pathological changes occurred in the organs. The biochemical parameters for the liver and kidney were significantly up-regulated, and there was 6PPDQ accumulation in the liver and lungs.
2024	[96]	Combined exhaust gases, benzo[a]pyrene, and tyre particles.	Combined emissions’ toxicity in mice cell macrophages in vitro.		Laboratory by cryo-grinding commercial tyres.	No cytotoxicity or ROS production. B[a]P caused an inflammatory response, and tyre particles caused a size-dependent response; exhaust gases did not. For the larger particles, there was a disproportionate increase in TNF-α production, suggesting a synergistic effect.
2024	[65]	Ground tyre particles and actual tyre and road wear particles.	In vitro toxicity of ground TP and actual TRWP emissions in mice cells.		TPs: cryogenic grinding of a tyre. TRWPs: on-road sample collection from a vehicle.	No cytotoxicity or oxidative stress. Both particles induced a concentration-dependent proinflammatory response, but the TNF-α production was slightly higher for TRWPs.
(b)
Year	Ref.	Source	Species	Parameters Examined	Testing Conditions	Main Conclusions
2005	[56]	Tyre debris leachates.	R. subcapitata (microalga), D. magna (crustacean), and X. laevis (frog, amphibian).	Tyre debris leachates’ toxicity in vivo and in vitro (FETAX) in aquatic organisms.	Laboratory by rotating a new tyre against a steel brush.	The results showed strong dose-dependent growth inhibition in R. subcapitata and death rate in D. manga and X. laevis.
2007	[97]	Tyre debris organic extracts.	Xenopus (frog).	Tyre debris organic extracts’ embryotoxicity in frogs (FETAX test).	Laboratory by cryo-fracturing tyre scraps.	The NOEC was at 50 mg/L, mortality and percentage of malformed larvae was increased at 80 mg/L, and the probit analysis gave a 144.6 mg/L TC50. There were mostly ocular malformations and severe vacuolisation and necrosis in the liver and axial musculature.
2009	[98]	Tyre debris (Zn2+) and ZnCl2.	R. sylvatica (frog).	Tyre debris (Zn) toxicology in frog larvae (effects on metamorphosis).	Laboratory from used tyre scraps.	Both the exposures caused an increase in the time for larvae to complete metamorphosis. The increase metamorphosis time caused a decrease in the subject’s mass.
2009	[99]	Sequential tyre powder leachates.	D. magna, C. dubia (crustaceans), P. subcapitata (algae), and D. rerio eggs (fish).	Toxicity of tyre leachates in vivo and in vitro in aquatic life.	Laboratory by abrading tyres with a rasp and then leachating the powder (1 slightly worn and 2 heavily worn tyres).	The slightly worn tyre was shown to cause the most significant toxic response. The sequential leachates were also shown to be much less toxic than the first one. Toxicology was mostly attributed to zinc.
2010	[100]	Tyre particle leachates.	U. lactuca (marine macroalga).	In vivo toxicity to macroalgae and Zn release.	Abrasion of 20 end-of-life car tyres with stainless steel file.	Exposure to increasing concentrations of leachate resulted in a non-linear reduction in the efficiency of photochemical energy conversion and an increase in Zn accumulation.
2011	[101]	Tyre and road wear particle sediment elutriate and leachate.	P. subcapitata (algae), D. manga (crustacean), and P. promelas (fish).	In vivo acute toxicity of tyre and road particles to aquatic life.	Road simulator laboratory in an interior drum testing system containing actual asphalt pavement in cassettes (1 summer and 1 winter silica-based tyre, 1 carbon-black-based summer tyre).	No concentration response was observed, but some toxicity was observed at higher incubation temperatures.
2013	[102]	Tyre and road wear particle sediments and elutriates.	C. dilutus (holometabolous aquatic insect), H. azteca, C. dubia (crustaceans), and Pimephales promelas (fish).	Chronic toxicity of tyre and road wear particles in vivo in water- and sediment-dwelling organisms.	Road simulator laboratory in an interior drum testing system containing actual asphalt pavement in cassettes (1 summer, 1 winter silica-based tyre, 1 carbon-black-based summer tyre).	The sediments caused a mild growth inhibition in C. dilutes but had no effect on growth or reproduction in H. azteca. The elutriates caused a slight diminished survival in larval P. promelas but had no effect on growth or reproduction in C. dubia.
2018	[73]	Tyre particles.	G. pulex, A. aquaticus (crustaceans), L. variegatus, and Tubifex spp. (worms).	Tyre tread particle toxicity in freshwater invertebrates.	Laboratory through cryo-grinding of a tyre tread.	The results showed no effect on the survival, growth, and feeding rate of G. pulex and A. aquaticus, the survival and growth of Tubifex spp., and the number of worms and growth of L. variegatus.
2019	[103]	Tyre wear particles and tyre leachates.	H. azteca (crustacean).	Acute and long-term toxicology of tyre wear particles and leachates in freshwater organisms.	Laboratory by surface abrasion of one road-worn tyre for tyre wear particles; TWPs were used for leaching.	Mortality, reproductive output, and net growth were significantly affected at higher concentrations. Tyre wear particles showed a typical concentration–response pattern, while leachates did not.
2020	[83]	Tyre rubber leachates compared to other plastics.	R. subcapitata, S. costatum (microalgae), and M. galloprovincialis (mussel).	Tyre wear toxicity in aquatic life (freshwater and marine).	Laboratory using tyre-derived granulate reference materials.	Tyre leachates were found to be amongst the most toxic samples tested. They caused the inhibition of algal growth, significant down-regulation of gamete fertilisation, and inhibition of normal embryonic development in mussels.
2020	[77]	Tyre particle leachates.	P. promelas (fish).	Tyre leachates’ in vivo toxicity in fish comparing different physical stressors (temp., UV, CO2, mechanical stress).	Laboratory by manually cutting small pieces from new tyres.	The results showed that the variations in temperature and mechanical stress caused a significant change in toxicity, whereas UV and CO2 exposure caused milder effects.
2020	[104]	Tyre particles (crumb rubber).	F. heteroclitus and P. promelas (fish).	In vivo toxicity through 7-day static renewal exposure.	Crumb rubber from passenger and truck tyres.	Highest concentration tested caused partial mortality in P. promelas. Bile fluorescence and EROD assay indicated that there was a presence of PAHs.
2021	[105]	Pristine tyre particles and leachates compared to road-worn TWPs and leachates.	H. azteca (crustacean).	Tyre wear particle suspensions’ and leachates’ toxicity in aquatic life.	Laboratory by grinding two tyres (new and used).	New TP suspensions were more toxic than those of worn TPs; however, leachates were equally toxic. New TP suspensions showed no significant effects on mortality and reproduction, but growth was significantly reduced at the highest concentration tested.
2021	[76]	Tyre wear particle leachates.	Coho and Chum salmon (fish).	Tyre leachates’ in vivo toxicity in coho and chum salmon.	Laboratory by grinding nine unique tyres (2 new, 7 used).	Coho salmon displayed significant behavioural changes, mortality, and changes in blood parameters after exposure. Chum salmon was unaffected by exposure.
2021	[84]	Tyre wear particles compared to polystyrene microplastics.	C. riparius (midge).	Tyre wear particles’ in vivo toxicity in a midge.	Laboratory through cryo-grinding of an end-of-life tyre.	Several genes encoding heat shock proteins were overexpressed, and genes coding for manganese superoxide dismutase and for the FK506-binding protein were altered. Microplastics caused cellular stress which led to some gene alterations but did not cause any mortality.
2022	[85]	Tyre wear particles compared to PET particles (leachates).	Freshwater bacterial community.	Tyre wear microbiological effects on freshwater ecosystems.	Laboratory by shredding of a used tyre.	TWPs caused increased bacterial growth and the creation of a biofilm that was much less diverse compared to PET.
2022	[106]	Tyre wear particles and leachates.	A. bahia (crustacean) and M. beryllina (fish).	Tyre particles’ and leachates’ toxicity and behavioural changes in aquatic organisms.	Laboratory through cryo-milling of a tyre tread.	TWPs caused significant alterations in swimming behaviours, a concentration-dependent reduction in growth after exposure to microparticles for both species, and a reduction in growth after exposure to nanoparticles for M. beryllina.
2022	[107]	Tyre particles and leachates.	D. rerio (fish) and D. manga (crustacean).	Toxicity of micro and nano tyre particles and leachates in freshwater organisms.	Laboratory through cryo-milling of a tyre.	Nano tyre particles and leachates caused an increased mortality and sublethal malformations, but micro tyre particles did not.
2022	[108]	6PPD and 6PPDQ.	D. rerio (fish).	6PPD and 6PPDQ toxicity in fish larvae.	-	6PPDQ was found to be toxic, but no significant mortality was observed. There was a dose-dependent reduction in swimming performance in both compounds.
2022	[109]	Tyre wear (crumb rubber) leachates.	F. heteroclitus (fish).	Tyre leachates’ in vivo toxicity in fish with episodic exposure.	Crumb rubber.	CYP1A was up-regulated in gills, intestine, and liver, and there was a dose-dependent increase in bile fluorescence. There was increased 8-OHdG and total GSH and decreased MDA production.
2022	[110]	End-of-life tyre-derived granules and powder.	P. subcapitata (algae), D. manga (crustacean), and D. rerio (fish).	Tyre granules’ and powder suspensions’ acute and chronic in vivo toxicity in freshwater organisms.	ELT granules and powder from a tyre recycling plant.	Acute tests showed EC50 of over 100 μg/L for D. magna and D. rerio. Chronic tests showed an LOEC of 9.8 μg/L for D. magna and 10 μg/L for D. rerio. There were significant effects on the cell density of P. subcapitata at 100 μg/L.
2022	[86]	Tyre particles compared to polyester fibres.	D. manga (crustacean), H. azteca, A. aquaticus (crustaceans), and L. variegatus (worm).	Tyre particles’ acute and chronic in vivo toxicity in freshwater invertebrates.	End-of-life tyre granules collected from manufacturer and milled.	There was no effect on mobility after acute exposure, but there was an effect on survival and reproduction after chronic exposure in D. magna. There was no effect on survival for the other species; however, there was significant ingestion.
2022	[111]	Tyre wear particle leachates.	B. plicatilis (rotifer).	Tyre wear particle leachate in vivo toxicity in a euryhaline rotifer.	Laboratory from a tyre simulator (drum).	Acute toxicity was observed. Reproduction and population growth were decreased, and oxidative stress was induced after exposure. There was an increase in antioxidant enzymes and an induction of transcriptional dysfunction of 1082 DEGs.
2022	[74]	Tyre wear particles (microrubber).	C. riparius (midge) and L. variegatus (worm).	Tyre wear particle in vivo toxicity in freshwater sediment organisms.	Laboratory through cryo-grinding of an end-of-life tyre.	The results showed no significant effects on growth, survival, or reproduction.
2022	[112]	Tyre wear particles.	S. plana (mollusc) and H. diversicolor (worm).	Tyre wear particle in vivo toxicity in estuarine invertebrates.	Laboratory through grinding of used tyres (with liquid nitrogen).	S. plana had reductions in burial and feeding rates. Protein concentration and total energy content were affected. Total glutathione in H. diversicolor and lipid peroxidation in S. plana were changed.
2022	[113]	Tyre particles and leachates.	T. japonicus (crustacean).	In vivo toxicity to marine copepod.	Waste tyre crushing factory from heavy-duty trucks.	LC50 was 771.4 mg/L for particles and 5.34 g/L for leachates. Zn was identified as the main cause of the toxicity.
2023	[75]	6PPDQ.	Hexagenia spp. (insect), D. magna (crustacean), Planorbella pilsbryi (mollusc), and M. nervosa (mussel).	6PPDQ toxicity in vivo for four freshwater invertebrate species.	-	For all four species, the highest concentrations tested did not result in significant mortality.
2023	[114]	6PPDQ and DTBBA.	Brachionus koreanus (marine rotifer).	6PPDQ and DTBBA toxicity in a marine rotifer.	-	Only moderate toxicity was observed for 6PPQ; however, there was a significant response in population growth and fecundity for DTBBA.
2024	[78]	Aged tyre wear particles and leachates.	P. tricornutum (microalgae).	Aged TWPs’ and leachates’ in vivo toxicity in microalgae.	Laboratory by grating a used tyre.	Growth of microalgae was inhibited. Chla and Car increased over time. No significant effect on SOD and MDA. Overall, whole particles were more toxic than leachates and aged particles more than virgin ones.
2024	[115]	Tyre additives (leachates).	D. rerio (fish).	Tyre additives’ in vivo toxicity in fish (ophthalmological effects).	-	MBT (2-mercaptobenzothiazole) was found to be one of the main toxic compounds, as it caused a significant decrease in eye size and cell density. Many gene expressions were also changed.
2024	[116]	Pristine and aged TWPs and four bisphenols.	T. japonicus (crustacean).	In vivo toxicity of combined pristine and aged TWPs and four bisphenols in a crustacean.	Laboratory by grinding end-of-life tyres.	TWPs increased the toxicity of BPA and BPF and decreased the toxicity of BPAF. For BPS, there was a synergistic toxic effect with p-TWPs but a slightly antagonistic effect with a-TWPs.
2024	[117]	TWP leachates.	R. salina (microalga) and D. rerio (zebrafish) and U-2 OS cells and CALUX assay.	In vivo (R. salina and D. rerio) and in vitro (U-2 OS cells and CALUX assay) toxicity of TWP leachates.	Laboratory by milling unused tyres.	Leachates inhibited algal growth and induced embryotoxicity, pigment alterations, and behavioural changes in D. rerio. In vitro testing showed pro-apoptotic changes and endocrine-disrupting potential.
2024	[118]	Tyre wear particles.	D. rerio (zebrafish).	In vivo toxicity in the gills, liver, and gut and accumulation and depuration of TWPs.	Laboratory by grinding mini-car tyre scraps.	There was a dose-dependent TWP accumulation in the gills and gut for a long time, and oxidative stress was induced in the gills and liver. There were dose-dependent up- and down-regulations of metabolic processes by the liver.
(c)
Year	Ref.	Source	Species	Parameters Examined	Testing Conditions	Main Conclusions
2011	[119]	Crumb rubber.	13 soil nematodes (mainly Helicotylenchus) (worm).	Crumb rubber toxicity and effects on nematodes and soil.	-	After exposure, the plant parasite and omnivorous nematode population decreased, and the predatory nematode population increased. Soil bulk density and pH were decreased, and soil moisture was increased.
2018	[120]	Aged crumb rubber.	E. fetida (worm).	Aged crumb rubber toxicity to earthworms.	-	Exposure did not reduce earthworm body weight, but it reduced survival time during a stress test. Microbial respiration rates were not impacted.
2020	[121]	Tyre tread particles.	E. crypticus (worm).	Tyre tread particle effects on soil fauna and worm gut microbiota.	Laboratory by grating a used tyre.	Exposure caused a decrease in survival and reproduction and disrupted the microbiota of the worm gut and the soil.
2021	[122]	Tyre tread particles.	E. fetida (worm).	Tyre particles’ effect on earthworms (bioaccumulation of heavy metals and oxidative stress).	Laboratory by cryo-grinding a used tyre tread.	Higher concentrations caused oxidative stress, as well as increased catalase and peroxidase activity and lipid peroxidation levels, and reduced activity of SOD and GST.
2021	[123]	Tyre particles.	E. crypticus (worm), F. candida (springtail), and P. scaber (crustacean).	Tyre particles’ toxicology when spiked in soil or food.	Laboratory through cryo-milling end-of-life tyres.	In soil, at high concentrations, there was a decrease in F. candida reproduction and survival and the AChE activity of P. scaber. In food, at high concentrations, F. candida survival was reduced.
2021	[124]	Tyre particles.	Soil bacterial community.	Tyre particles’ effect on bacterial community when spiked into soil.	Laboratory by grating a used tyre.	Different ARGs were found in the samples spiked with tyre particles compared to the control, and the amounts were significantly increased when placed under stress from heavy metals and antibiotics.
2021	[125]	Tyre wear particles.	Sinapis alba L. and Lepidium sativum L. (plants).	Tyre particle phytotoxicity (biological activity, CO2, germination index).	-	Soils with 50 and 75% tyre particles showed decreased biological activity and CO2-C emissions. There was a subchronical phytotoxicity and a lower germination index compared to the control.
2021	[126]	Tyre wear particles.	C. elegans (worm).	Tyre particle time-dependent toxicity (soil pre-incubation and exposure time).	Laboratory by grinding a used tyre.	The incubation increased the toxicity of the tyre wear particles, and the lifetime of the organisms was reduced faster in the treated groups, especially with lifetime exposure.
2022	[127]	Tyre wear particles compared to polyester fibres and natural particles.	P. scaber (terrestrial crustacean).	Tyre particle time-dependent toxicity (immune system).	Laboratory by cryo-milling used tyre scraps.	After 4 days of exposure, the total number of haemocytes was decreased, and the proportions of different haemocytes were altered. After 7 days of exposure, there was an increase in superoxide dismutase activity and metabolic activity.
2022	[128]	Tyre wear particles from cars, bicycles, and e-scooters.	V. radiata (plant) and F. candida (springtail).	Tyre particle toxicity in soil organisms.	Laboratory by grating new tyre treads from 3 different personal mobility means.	Bicycles and scooters changed the soil’s bulk density and water holding capacity and reduced plant growth. Car tyre particles leached organic compounds and had severe effects on springtails.
2022	[129]	Microplastics (tyre particles and polystyrene).	E. andrei (worm).	Time-dependent in vivo toxicity in earthworms.	Bulk product of recycled tyres.	Only minor effects could be observed on a subcellular level at environmentally relevant concentrations.
2023	[130]	Tyre wear particles compared to polyester microfibres.	F. candida (springtail) and P. scaber (terrestrial crustacean).	Change in chlorpyrifos toxicity after exposure to tyre particles for soil arthropods.	Laboratory by cryo-milling used tyres.	The lethality of chlorpyrifos and its effects were reduced significantly after exposure.
2023	[131]	Tyre rubber and tyre leachates.	Unspecified soil microorganisms.	Toxicity in microorganisms.	Laboratory by grinding tyres.	No biodegradation in any sample, but the toxicity was higher when exposed to tyre shred particles. MTT test showed 28% inhibition of the viability for the samples with particles.
2023	[132]	6PPDQ.	C. elegans (worm).	In vivo toxicity of 6PPDQ in nematodes.	6PPDQ.	A total of 0.1–10 μg/L of 6-PPDQ caused several forms of abnormal locomotion behaviours and the neurodegeneration of D-type motor neurons at 10 μg/L.

summarises the studies that examined tyre particles in human/mammalian, aquatic, and terrestrial cells. More details about the studies can be found in Appendix A.

3.2.1. Human/Mammalian

In the studies conducted on mammalians, there was a big variety of sample collection and testing methodologies; hence, it can be difficult to compare results. Overall, three of the studies showed no significant effect caused by tyre wear particles [57,60,61]. A fourth in vitro study did not follow an up-to-standard methodology: only two strains of bacteria were used, even though, according to the OECD (Organisation for Economic Co-operation and Development) guidelines, at least five strains should be used [62]. Four studies showed mild effects [45,63,64,65], while the remaining seventeen studies showed significant toxicity. Out of the four studies that showed no effects, three were carried out in vitro. Additionally, out of these four studies, three of them were conducted using samples collected from a road simulator. However, as there were both in vitro studies and studies with road simulator samples that showed effects, no conclusion can be drawn regarding the non-reliability of any of these studies.

Some of the studies that used samples from a road simulator used different types of pavements during the sample creation, which affected the particles created, as a big proportion of the particles originated from the pavement surface. Overall, both granite and quartzite pavements were able to cause a toxicological response; however, granite caused a much stronger response. Granite caused higher cytokine release, and although both pavements induced similar lipid peroxidation and ROS formation, only granite induced nitrogen oxide (NO) production [66,67,68]. However, it should be noted that these studies used studded tyres, which are representative only of winter conditions in Scandinavian countries.

One study [69] used a three-dimensional (3D) human airway organoid made from human bronchial epithelial cells in order to assess the toxicity of tyre wear particles. This in vitro model can give a much more accurate overview of the real response that might occur in human airways when exposed to harmful particles. In this study, the samples were able to induce significant cytotoxicity at concentrations higher than 200 μg/mL, as well as significant ROS generation in a dose-dependent manner. An increase in oxidative stress-related and cytokine-related gene expressions including TNF-α, IL-6, and ccl2 was observed. Interestingly, the larger particles were found to be clustered around the epithelial cells, and the smaller particles were found inside the cells. This type of information was only observed because of the 3D model.

After the discovery that the by-products of tyre wear 6PPD (N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine) and 6PPDQ (N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine quinone) are toxic to coho salmon in 2020, much more research has been conducted in order to assess their toxicity to marine organisms, with over 25 papers about the effects of tyre wear particles on aquatic life being published since 2021. This will be discussed in the next section. In comparison, only 10 new papers about the health effects of tyres on mammalians have been published since 2021, to our knowledge. There are only two mammalian papers that studied the effects of 6PPD and 6PPDQ in mice [70,71]. Both papers found the substances to be bioaccumulative in the liver of mice in a dose-dependent manner. Exposure doses and frequencies seem to have a strong effect on the toxicity of these compounds [71], where a single injection did not cause any significant changes in the mice; however, repeated exposures were able to cause significant changes in biochemical parameters in multiple organs.

One more paper mentioned the existence of 6PPD and 6PPDQ in mammalians [72]. In this 2022 paper, the researchers conducted a biomonitoring study on humans, where they monitored the urine concentrations of these substances in three human populations: adults, children, and pregnant women. Both 6PPD and 6PPDQ were detected at frequencies between 60 and 100%, although the 6PPDQ concentrations were significantly higher. Interestingly, both concentrations were much higher in pregnant women compared to adults and children. After in vitro metabolic experiments, the results showed a rapid depletion of 6PPD by human liver microsomes, which could be responsible for the lower concentration of 6PPD. Considering all the research showing the extensive toxicological effects of these substances in other organisms, the fact that there are significant concentrations found in human urine should be a cause for worry, and it should push for more research to be conducted in this field.

3.2.2. Aquatic

Out of the thirty-three studies conducted on aquatic organisms, only three of them found no significant effects caused by tyre wear particles [73,74,75], while seven of them found some effect, and the remaining twenty-three showed significant effects. However, as these studies were all conducted in different organisms, it is hard to establish the particles’ toxicity.

As previously mentioned, a few years ago, there was a sudden interest in the tyre by-products 6PPD and 6PPDQ, after the discovery of their effects on coho salmon. Although at the beginning this finding was seen as a threat to all salmonid species, after more research, it was established that the toxicity primarily affected coho salmon. In fact, in a 2021 paper [76], the effects of tyre particles on coho and chum salmon were assessed, and the results showed that while coho salmon displayed significant behavioural changes, changes in blood parameters, and mortality, chum salmon seemed to be unaffected by exposure. This further confirms that the toxicity of tyre wear particles varies significantly between animals and even between species.

In another paper [77], the toxicity of tyre particles was assessed using samples that had been affected by different stressors. In this study, the researchers used two tyre samples that had been exposed to different levels of four physical stressors (temperature, mechanical stress, ultraviolet (UV) light, and CO₂). In the temperature experiments, there was no significant effect on time to hatch; however, hatching success decreased in the second-tyre experiments. Significant differences in heart rates were observed for different materials and different temperatures. Increasing deformities and shorter lengths were observed with increasing temperature, although the second tyre caused more severe deformities. Both mechanical stress and UV exposure caused very similar effects to temperature changes, where the effects were consistent between the two tyre types, although once again, the second tyre caused more severe effects. The effects of carbon dioxide were not significant enough to influence the leaching process. Overall, the results showed that the variations in temperature and mechanical stress caused a significant change in toxicity, whereas UV and CO₂ exposure caused milder effects. This study was able to show how the toxicity of particles can easily change through different stressors and, hence, why particles in real life, which are exposed to such stressors, can potentially be much more harmful than laboratory-made tyre wear particles.

Another paper in 2024 looked at different tyre particles after being put through stressors [78]. In this paper, the particles were exposed to UV irradiation and two different doses of K₂S₂O₈ in order to simulate short- and long-term aging. The results showed that aged particles exhibited enhanced toxicity towards microalgae, compared to virgin particles, because of their increased leaching potential and physiochemical damage. Moreover, the exposure to particles and their leachates altered the metabolic profiles, and especially UV- and high-dose-K₂S₂O₈-aged particles caused the biggest effect.

One study in 2009 assessed the toxicity of sequential tyre wear particle leachates [79]. Particles were placed in purified water multiple times in order to retrieve sequential leachates. The results showed that the sequential leachates were much less toxic than the original samples. This effect is most likely caused by the dilution that occurs after multiple leachings, making the final exposure concentrations much smaller and, hence, much less toxic.

3.2.3. Terrestrial Invertebrates

We found fourteen studies that were carried out on terrestrial organisms (one more on a bacterial community), mostly invertebrates and crustaceans. All of these studies used samples created in laboratories by cryo-milling or grating or crumb rubber. They all showed significant effects on the organisms studied; however, this could be because of higher exposures causing more toxicity. Overall, there are too few studies on terrestrial organisms, and more research needs to be carried out.

3.3. Species

Figure 3 summarises the number of studies that assessed the impact of tyre particles on different species. There is no clear tendency of a particular species being affected less or more by tyre particles.

3.4. Non-Exhaust vs. Other Particles

From all the papers analysed, sixteen of them compared non-exhaust emissions to other sources of particulate matter (Figure 4). Although it is hard to draw a direct comparison, as it is important to take the whole exposure pathway into consideration, it is, nevertheless, interesting to analyse the different sources of PM in order to have a more comprehensive view of the problem at hand.

Out of the papers on brake emissions, five of them discussed other sources of pollution [48,51,58,80,81]. Generally, it seems that brake particles are at least as toxic as the other sources tested, like urban dust and diesel and wood combustion. In a 2023 study, it was revealed that, although all the sources studied were found to be genotoxic, brake dust was the most cytotoxic [58]. Another study stated that brake pads caused the greatest decrease in cell viability among the sources tested [48].

From the papers on tyre emissions conducted on mammalians, seven of them [45,60,61,66,67,68,82] assessed other sources and compared them to tyre particles; interestingly, most of them were carried out using samples from studded tyres [60,66,67,68,82]. All concluded that the airway inflammatory and genotoxic potential of particles from studded tyres is at least equal, if not stronger, than that of other sources, more specifically, diesel particles. Most of the papers also agreed that subway particles caused a stronger response than any of the other sources tested. Lastly, one paper [61] showed no effect on the 3D intestine barrier model used in the study, for both tyre particles and the other microplastics tested.

On the other hand, out of the papers on the effects of tyre emissions on aquatic organisms, there were only four [83,84,85,86] that compared them with other sources, namely other microplastics and other non-traffic-related emissions. All four papers agreed that the tyre emissions had a more significant effect on the aquatic organisms tested, compared to polyethylene terephthalate (PET), polystyrene, polyester, polypropylene, and polyvinyl chloride (PVC).

Overall, most of the papers agreed that non-exhaust emissions are comparable to other sources like exhaust emissions and wood combustion. Some of the other sources assessed in these papers were poultry farms, polyester fibres, polystyrene microplastics, carbon nanotubes, diesel exhaust particles, coke dust, and pellet ashes. These findings prove that there should be a much more serious effort towards the regulation of non-exhaust emissions, considering that many of the other sources of pollution discussed, which are currently regulated in order to improve human health, are comparable to emissions from tyres and brakes, in term of toxicity.

3.5. Samples

It is crucial to highlight the lack of research on certain specific types of tyres and brakes.

Table A3. Summary of materials used in studies about health effects of brakes.

Ref.	Non-Asbestos Organic (NAO)	Low-Metallic	Semi-Metallic	Hybrid Vehicle	Prototype	Asbestos Containing	Heavy-Duty (All)	Not Specified
Mammalians	[48,80,87]	[46,48,80,87]	[48,80]	[80]	[46,47,52,59]	[53,54,55]	[51]	[49,50,58,59,88,89]
Aquatic								[81]

and

Table A4. Summary of materials used in studies about health effects of tyres on mammalians.

Ref.	Summer	Winter	All-Season	Winter Studded	Reference Material	6PPDQ and Extracts	Heavy-Duty	Not Specified
Mammalians	[57,62,94]	[57,68,82]	[94]	[60,66,67,68,82,92]	[43]	[70,71]	[61,93]	[43,44,45,56,63,64,65,69,90,91,93,95,96]
Aquatic	[101,102]	[101,102]			[83,104,109]	[75,108,114,115,116]	[104,113]	[56,73,74,76,77,78,84,85,86,97,98,99,100,103,105,106,107,110,111,112,116,117,118]
Terrestrial					[119,120,129]	[125,132]		[121,122,123,124,125,126,127,128,130,131]

in Appendix A show a clear lack of toxicological studies carried out on certain types of non-exhaust emissions. One of the most evident gaps in this review is the lack of research on heavy-duty vehicles, with only five studies using heavy-duty brakes or tyres in their toxicological assessment. Another noteworthy issue is the amount of research conducted on additives and extracts from tyres, as well as non-specified tyre and rubber materials, instead of real tyres.

There is a clear difference in composition between summer and winter tyres and, hence, in the composition of the particles released. This difference is even more significant for studded tyres which use tungsten carbide studs. This is why it is important to take into account the types of tyres used while assessing their abrasion. Moreover, heavy-duty tyres also have considerable differences in their composition. Similarly, brake systems vary immensely in their composition and functionality. There are non-negligible differences between markets and between uses.

4. Conclusions

This review paper gave an overview of the literature on the health effects of non-exhaust emissions and highlighted the gaps in the current research. Overall, most of the papers agree that particles from non-exhaust sources can cause adverse health effects. Almost all of the studies saw some toxicity; however, many of them showed that the effects can be extremely detrimental and should be a cause to worry. Some of the main issues seen were changes in inflammation and organ parameters like ROS, IL-8, and TNF-α, as well as issues in the lung and airway tissues. In some cases, the impact was attributed to heavy metals like zinc and copper, PAHs, 6PPDQ, dithiobisbenzanilide (DTBBA), 2-mercaptobenzothiazole (MBT), and other chemicals, however, without being able to establish a causal relationship. Studies that compared non-exhaust particles with other particles (subway, urban environment, and exhaust particles for mammalians or PET/PS for aquatic cells) found that tyres and brakes are in general more harmful.

Even though there are many toxicological studies in the literature, there is a need for further research in order to have a full picture of the real effects of tyre and brake emissions. One of the main concerns with the current literature is that in many cases the samples used in the toxicological assessment were created in the laboratory in a way that is not representative of real-life emissions. This means that the results of these studies are not necessarily representative of the real health effects caused to humans when exposed to such particles. It is important to understand at what levels non-exhaust particles become (or stop being) harmful and compare those with measured concentrations in various environmental compartments. One important thing to note is the lack of toxicological research for certain types of non-exhaust emissions, like those derived from heavy-duty vehicles. Another significant issue in this area is the lack of epidemiological studies, which are the golden standard when it comes to health research.