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.

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.

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.

Modulation of key elements of the Wnt pathway by apple polyphenols.

Glycogen synthase kinase-3beta (GSK3beta) is one of the key elements of the Wnt pathway involved in the regulation of beta-catenin homeostasis. The inhibition of GSK3beta kinase activity might lead to the onset of beta-catenin/TCF/LEF-mediated gene transcription, representing a potentially mitogenic stimulus. Apple polyphenols have been shown to mediate several biological effects that might be of interest with respect to chemoprevention. The objective of the study was to elucidate whether apple polyphenols also modulate key elements of the Wnt pathway, an effect that might limit the usefulness of these compounds for the prevention of carcinogenesis. A polyphenol-rich apple juice extract (AE02) was found to effectively inhibit the kinase activity of GSK3beta, immunoprecipitated from HT29 cells. Treatment of HT29 cells with AE02 for 24 h resulted in a concentration-dependent decrease of the cellular GSK3beta kinase activity. The inhibition of the kinase activity in HT29 cells was observed at polyphenol concentrations corresponding to the concentration of the constituents in the original apple juice. The apple characteristic dihydrochalcone glycoside phloridzin was found to be inactive up to 500 muM. The free aglycon phloretin as well as the flavonol quercetin effectively inhibited isolated GSK3beta, but did not affect the respective kinase activity within HT29 cells. In accordance with the inhibition of GSK3beta kinase activity by AE02, treatment of HT29 cells resulted in a significant decrease of phosphorylated beta-catenin. However, the total intracellular beta-catenin level was also found to be diminished, indicating that the interference of the apple constituents with GSK3beta was not associated with a stabilization of beta-catenin in HT29 cells. In line with these results, TCF/LEF-mediated gene transcription remained unaffected by treatment with AE02 as shown in a reporter gene approach. We can assume from the results that at consumer-relevant concentrations apple polyphenols do not mediate growth-stimulating effects in HT29 cells via the Wnt pathway.

Impact of quercetin and EGCG on key elements of the Wnt pathway in human colon carcinoma cells.

The flavonoids quercetin (QUE) and (-)-epigallocatechin-3-gallate (EGCG) are discussed as potential chemopreventive food constituents. Both compounds have been shown to affect a spectrum of different cellular signaling pathways. Glycogen synthase kinase-3beta (GSK3beta) is one of the key elements of the Wnt pathway, governing beta-catenin homeostasis. The inhibition of GSK3 kinase activity might lead to the onset of beta-catenin/TCF/LEF-mediated gene transcription, representing a potentially mitogenic stimulus. The aim of the study was to elucidate whether QUE and EGCG possibly mediate undesired proliferative stimuli in human colon carcinoma cells by interference with the Wnt pathway. In HT29 cells QUE did not inhibit the activity of GSK3alpha and -beta, measured as phosphorylation at Ser21 and Ser9, respectively. In accordance, QUE did not substantially affect beta-catenin homeostasis. In a reporter gene assay QUE was found to act as a weak inductor of T-cell factor/lymphoid enhancer factor (TCF/LEF) mediated luciferase expression, which was, however, not associated with a stimulation of cell growth. Treatment of HT29 cells with EGCG led to a potent inhibition of GSK3alpha and -beta activity. Subsequently, the amount of phosphorylated beta-catenin was diminished in a concentration-dependent manner. Concomitantly, the overall amount of beta-catenin was decreased to a similar extent, which might result from a downregulation of beta-catenin neogenesis, indicated by reduced levels of beta-catenin mRNA. In accordance, no induction of TCF/LEF-mediated luciferase expression was observed. In conclusion, the results allow the assumption that QUE and EGCG do not mediate proliferative stimuli in HT29 cells by interference with key elements of the Wnt pathway.

Inhibitors of the epidermal growth factor receptor in apple juice extract.

The polyphenol-rich extract of a consumer-relevant apple juice blend was found to potently inhibit the growth of the human colon cancer cell line HT29 in vitro. The epidermal growth factor receptor (EGFR) and its subsequent signaling cascade play an important role in the regulation of cell proliferation in HT29 cells. The protein tyrosine kinase activity of an EGFR preparation was effectively inhibited by the polyphenol-rich apple juice extract. Treatment of intact cells with this extract resulted in the suppression of the subsequent mitogen-activated protein kinase cascade. Amongst the so far identified apple juice constituents, the proanthocyanidins B1 and B2 as well as quercetin-3-glc (isoquercitrin) and quercetin-3-gal (hyperoside) were found to possess substantial EGFR-inhibitory properties. However, as to be expected from the final concentration of these potential EGFR inhibitors in the original polyphenol-rich extract, a synthetic mixture of the apple juice constituents identified and available so far, including both proanthocyanidins and the quercetin glycosides, showed only marginal inhibitory effects on the EGFR. These results permit the assumption that yet unknown constituents contribute substantially to the potent EGFR-inhibitory properties of polyphenol-rich apple juice extract. In summary, the polyphenol composition of apple juice possesses promising growth-inhibitory properties, affecting proliferation-associated signaling cascades in colon tumor cells.