Response to letter to the editor

We read with interest the statements of Burghardt et al. concerning our paper “Induction of chromosomal damage in exfoliated buccal and nasal cells of road markers” (Wultsch et al. 2019).

We found a significant increase of chromosomal damage (cells with micronuclei, MN) in buccal and nasal cells of road markers and postulated that components of the stain (reflecting pearls and/or different chemicals) may cause this effect. Burghardt et al. stated in their comments that the size of the particles (200 µm) is too large to make them inhalable and that they consist of glass and that SiO₂ in crystalline form is fully bound. Furthermore, they mention that even glass beads with diameters smaller than 1.65 µm do not cause silicosis. The description of the paint materials which are generally used for road marking mention often that the particle sizes vary between 50 and 200 µm, and it cannot be excluded that smaller particles are contained as impurities. There is clear experimental evidence that small glass particles cause damage of genetic material in cultured rodent and human-derived cells (Casey 1983; Ong et al. 1997; Pelin, Kivipensas, and Linnainmaa 1995; Zhong, Ong, and Whong 1997) and also in human-derived alveolar epithelial cells (Rapisarda et al. 2015). The fact that small particles were not found to cause silicosis does not argue against the assumption that they cause genetic damage in cells of the upper respiratory tract which may lead to cancer.

The authors provided also some statements concerning the chemicals that are used. Based on theoretical consideration they argue that dibenzoyl peroxide cannot be inhaled or that the amounts are extremely low and therefore neglectable. Furthermore, they state that the levels of other chemicals do not exceed the current regulations. These statements do not argue against the induction of DNA damage in the workers via exposure to the paints. In many cases exposure limits defined by health authorities had to be redefined. The strong smell of the air which is inhaled by the workers during the application of the paints is strongly indicative for chemical exposure. Whether the chemicals contained in the paint materials themselves or reaction products which formed during the application is not of high relevance in regard to the question if they cause DNA damage or not.

Burghardt et al. also criticize that the exposure of the workers in our study was not quantified. This is indeed a shortcoming of our study. To compensate for it, a control group was used which was exposed to ambient air in the same city at the same time period when the sampling of the road markers was realized. This excludes to a certain extent the confounding by the general air composition. Notably, the extent of MN formation increased in the markers as a function of the working duration.

The authors also mentioned that the groups were not perfectly matched, i.e. the controls were on average 5 years younger. Note in this context that such a narrow difference in the age range is unlikely to affect the results of MN experiments with buccal cells (Bonassi et al. 2011; Ferraz et al. 2016). In addition, age was included among the covariates in the analyses and would adjust for residual effects of age.

Burghardt et al. also mention that smoking and alcohol consumption have an impact on the results. The number of smokers in the exposed group was indeed higher than in the control but the number of cigarettes smoked per day and the duration of smoking (not shown in Table 1) did not differ significantly. It is well known that only smoking of ≥40 cigarettes per day (Bonassi et al. 2011) or smoking of ≥25/day cigarettes without filter (Nersesyan et al. 2011) influences MN formation in buccal cells. Furthermore, the influence of smoking on MN rates in buccal cells of workers exposed to different genotoxins was inconclusive (Diler and Celik 2011; Diler and Ergene 2010; Javed and Ghani 2017; Khan et al. 2010; Singaravelu and Sellappa 2015). In our study, we did not find any influence of smoking on MN formation in the exposed workers (i.e. the p value for buccal cells was 0.27 and that for nasal cells 0.55). Also, in regard to alcohol consumption, the difference between the groups was not statistically significant (p value with Yate’s corrections is 0.17) (see Supplementary Table 2). The influence of smoking on MN formation in nasal cell is not well studied (Knasmueller et al. 2011) but there are several investigations indicating that smoking does not increase the MN levels in nasal cells of workers exposed to carcinogenic heavy metals (Burgaz et al. 2002; Kiilunen et al. 1997).

The statements of the authors are more or less entirely based on theoretical considerations and do not contradict our field-experimental observations. The article which we published describes the findings of a pilot study which is, according to our knowledge, the first investigation which indicates that road markers have increased health risks due to chromosomal damage in cells of upper respiratory tract. In order to confirm our findings well-designed studies should be realized along with laboratory experiments with different materials. Theoretical considerations will not substantially contribute to a better risk assessment of the workers and they can also not prove that the results of our study are irrelevant.