Comment on Balwierz et al. Potential Carcinogens in Makeup Cosmetics. Int. J. Environ. Res. Public Health 2023, 20, 4780.

We read with interest the article by Balwierz et al. [...].

Toxicokinetic study following intratracheal instillation or oral gavage of two [7Be]-tagged carbon black samples.

BACKGROUND: The toxicokinetic behaviour of nanostructured particles following pulmonary or oral deposition is of great scientific interest. In this toxicokinetic study, following the general principles of OECD TG 417, the systemic availability of carbon black, a nanostructured material consisting of agglomerated aggregates was characterised. METHODS: Each of two grades of beryllium-7 labelled carbon black (Monarch® 1000, oxidized and Printex® 90; untreated) was administered either intratracheally or orally to adult rats. Independent of route, rats received a single dose of approximately 0.3 mg radiolabelled carbon black. A total of 12 rats were treated per grade and per exposure route: 4 females each for feces/urine/organs and serial blood kinetics; 4 males for organs. At necropsy, the complete suite of organs was analysed for females, but only the lungs, liver, kidney, reproductive organs for males. RESULTS: In the pulmonarily exposed animals, 7Be-Monarch® 1000 and 7Be-Printex® 90 was detected in feces in the first 3 days after treatment at significant levels, i.e. 17.6% and 8.2%, respectively. In urine, small percentages of 6.7% and 0.4% were observed, respectively. In blood, radioactivity, representative of carbon black was within the background noise of the measurement method. At necropsy, 20 days post-instillation, both test items were practically exclusively found in lungs (75.1% and 91.0%, respectively) and in very small amounts (approximately 0.5%) in the lung-associated lymph nodes (LALN). In the other organs/tissues the test item was not detectable. BAL analyses indicated that carbon black particles were completely engulfed by alveolar macrophages. In orally exposed animals, 98% (7Be-Monarch® 1000) and 99% (7Be-Printex® 90) of the measured radioactivity was detected in feces. Excretion was complete within the first 3 days following treatment. 1.3% and 0.5% of measured activity was attributable to urine in animals that received 7Be-Monarch® 1000 and 7Be-Printex® 90, respectively. Radioactivity was absent in blood and other organs and tissues. CONCLUSION: Radioactivity, representative of carbon black, was not detected beyond the experimentally defined limit of quantitation systemically after deposition in lungs or stomach in rats. Under these experimental conditions, the two CB samples were not shown to translocate beyond the lung or the GI tract into the blood compartment.

Comment on Saber et al. (2019), "Commentary: the chronic inhalation study in rats for assessing lung cancer risk may be better than its reputation".

In their Commentary Saber et al. (Part Fibre Toxicol 16: 44, 2019) argue that chronic inhalation studies in rats can be used for assessing the lung cancer risk of insoluble nanomaterials. The authors make several significant errors in their interpretation and representation of the underlying science. In this Letter to the Editor we discuss these inaccuracies to correct the scientific record. When the science is recounted accurately it does not support Saber et al's statements and conclusions.

Evaluating the evidence on genotoxicity and reproductive toxicity of carbon black: a critical review.

Carbon black is produced industrially by the partial combustion or thermal decomposition of gaseous or liquid hydrocarbons under controlled conditions. It is considered a poorly soluble, low toxicity (PSLT) particle. Recently, results from a number of published studies have suggested that carbon black may be directly genotoxic, and that it may also cause reproductive toxicity. Here, we review the evidence from these studies to determine whether carbon black is likely to act as a primary genotoxicant or reproductive toxicant in humans. For the genotoxicity endpoint, the available evidence clearly shows that carbon black does not directly interact with DNA. However, the study results are consistent with the mechanism that, at high enough concentrations, carbon black causes inflammation and oxidative stress in the lung leading to mutations, which is a secondary genotoxic mechanism. For the reproductive toxicity endpoint for carbon black, to date, there are various lung instillation studies and one short-term inhalation study that evaluated a selected number of reproduction endpoints (e.g. gestational and litter parameters) as well as other general endpoints (e.g. gene expression, neurofunction, DNA damage); usually at one time point or using a single dose. It is possible that some of the adverse effects observed in these studies may be the result of non-specific inflammatory effects caused by high exposure doses. An oral gavage study reported no adverse reproductive or developmental effects at the highest dose tested. The overall weight of evidence indicates that carbon black should not be considered a direct genotoxicant or reproductive toxicant.

Evaluation of pulmonary and systemic toxicity following lung exposure to graphite nanoplates: a member of the graphene-based nanomaterial family.

BACKGROUND: Graphene, a monolayer of carbon, is an engineered nanomaterial (ENM) with physical and chemical properties that may offer application advantages over other carbonaceous ENMs, such as carbon nanotubes (CNT). The goal of this study was to comparatively assess pulmonary and systemic toxicity of graphite nanoplates, a member of the graphene-based nanomaterial family, with respect to nanoplate size. METHODS: Three sizes of graphite nanoplates [20 µm lateral (Gr20), 5 µm lateral (Gr5), and <2 µm lateral (Gr1)] ranging from 8-25 nm in thickness were characterized for difference in surface area, structure,, zeta potential, and agglomeration in dispersion medium, the vehicle for in vivo studies. Mice were exposed by pharyngeal aspiration to these 3 sizes of graphite nanoplates at doses of 4 or 40 µg/mouse, or to carbon black (CB) as a carbonaceous control material. At 4 h, 1 day, 7 days, 1 month, and 2 months post-exposure, bronchoalveolar lavage was performed to collect fluid and cells for analysis of lung injury and inflammation. Particle clearance, histopathology and gene expression in lung tissue were evaluated. In addition, protein levels and gene expression were measured in blood, heart, aorta and liver to assess systemic responses. RESULTS: All Gr samples were found to be similarly composed of two graphite structures and agglomerated to varying degrees in DM in proportion to the lateral dimension. Surface area for Gr1 was approximately 7-fold greater than Gr5 and Gr20, but was less reactive reactive per m(2). At the low dose, none of the Gr materials induced toxicity. At the high dose, Gr20 and Gr5 exposure increased indices of lung inflammation and injury in lavage fluid and tissue gene expression to a greater degree and duration than Gr1 and CB. Gr5 and Gr20 showed no or minimal lung epithelial hypertrophy and hyperplasia, and no development of fibrosis by 2 months post-exposure. In addition, the aorta and liver inflammatory and acute phase genes were transiently elevated in Gr5 and Gr20, relative to Gr1. CONCLUSIONS: Pulmonary and systemic toxicity of graphite nanoplates may be dependent on lateral size and/or surface reactivity, with the graphite nanoplates > 5 µm laterally inducing greater toxicity which peaked at the early time points post-exposure relative to the 1-2 µm graphite nanoplate.

Translational toxicology in setting occupational exposure limits for dusts and hazard classification - a critical evaluation of a recent approach to translate dust overload findings from rats to humans.

BACKGROUND: We analyze the scientific basis and methodology used by the German MAK Commission in their recommendations for exposure limits and carcinogen classification of "granular biopersistent particles without known specific toxicity" (GBS). These recommendations are under review at the European Union level. We examine the scientific assumptions in an attempt to reproduce the results. MAK's human equivalent concentrations (HECs) are based on a particle mass and on a volumetric model in which results from rat inhalation studies are translated to derive occupational exposure limits (OELs) and a carcinogen classification. METHODS: We followed the methods as proposed by the MAK Commission and Pauluhn 2011. We also examined key assumptions in the metrics, such as surface area of the human lung, deposition fractions of inhaled dusts, human clearance rates; and risk of lung cancer among workers, presumed to have some potential for lung overload, the physiological condition in rats associated with an increase in lung cancer risk. RESULTS: The MAK recommendations on exposure limits for GBS have numerous incorrect assumptions that adversely affect the final results. The procedures to derive the respirable occupational exposure limit (OEL) could not be reproduced, a finding raising considerable scientific uncertainty about the reliability of the recommendations. Moreover, the scientific basis of using the rat model is confounded by the fact that rats and humans show different cellular responses to inhaled particles as demonstrated by bronchoalveolar lavage (BAL) studies in both species. CONCLUSION: Classifying all GBS as carcinogenic to humans based on rat inhalation studies in which lung overload leads to chronic inflammation and cancer is inappropriate. Studies of workers, who have been exposed to relevant levels of dust, have not indicated an increase in lung cancer risk. Using the methods proposed by the MAK, we were unable to reproduce the OEL for GBS recommended by the Commission, but identified substantial errors in the models. Considerable shortcomings in the use of lung surface area, clearance rates, deposition fractions; as well as using the mass and volumetric metrics as opposed to the particle surface area metric limit the scientific reliability of the proposed GBS OEL and carcinogen classification.

Does carbon black disaggregate in lung fluid? A critical assessment.

Carbon black is an industrially produced particulate form of nearly pure elemental carbon. The basic building blocks of carbon black are (1) primary particles, minute pieces of matter with defined physical boundaries; (2) aggregates, collections of strongly bound or fused particles; and (3) agglomerates, collections of weakly bound aggregates. Industrial carbon black is produced within a closed reactor where the primary particles form aggregates, which become the indivisible entities of carbon black. These aggregates then form agglomerates, which are the typical form of carbon black in commerce. Carbon black is often used in in vitro and in vivo particle toxicology investigations as a reference nanoparticle. The toxicology studies often report the sizes of the primary particles but rarely the sizes of the aggregates or agglomerates. It appears in many cases that there is a limited understanding of the fact that carbon black typically does not exist as primary particles but instead exists as aggregates and agglomerates. Moreover, many toxicology studies manipulate carbon black particles in order to disperse them so that the form of carbon black used in these toxicology studies may be substantially different from the form that may be encountered in the workplace environment. Since the main exposure route for carbon black is inhalation, the question arose as to whether inhaled carbon black may deagglomerate or disaggregate to either smaller aggregates or primary particles when in contact with lung fluids. This question relates to the concern that there may be additional hazards of smaller particles, such as their ability to translocate to tissues and organs beyond the lung and the ability to pass through the blood-brain barrier. The purpose of this assessment is to review the existing literature for evidence as to whether carbon black deagglomerates or disaggregates into smaller aggregates or primary particles when in contact with lung fluid. On the basis of a review of the physical characteristics of commercial carbon black and various toxicology studies, it appears that commercially produced carbon black in contact with lung fluid is unlikely to deagglomerate or disaggregate into smaller aggregates or primary particles.