Ozone

Endpoint summary

Description of key information

Five research studies, in which the susceptibility of the skin to low ppm levels of ozone was studied, were retrieved from the scientific literature. It has been shown that short-term exposure of the murine skin to high levels of ozone (up to 10 ppm) caused damage in the stratum corneum (e.g. antioxidant depletion, lipid peroxidation), e.g. after 2-hour exposure to 1 ppm ozone as published by Weber et al. (1999) and Thiele et al. (1997a & b). Both research groups (Thiele et al., 2003, Weber et al., 1999) further showed that evidence for damage of the stratum corneum was already present and measureable by trans-epidermal water loss or significant depletion of vitamin C, glutathione and uric acid after 2-hour exposure to 1 ppm ozone. These findings are supported by He et al. (2006), studying the effects of 2-hour exposure to 0.8 ppm ozone on the levels of vitamin E, lipid peroxide, cutaneous bacteria and several key enzymes from the stratum corneum taken from human forearm skin samples. Visual observation and scoring revealed no skin irritation 30 minutes after ozone exposure. This study further provides supporting information that acute high ambient ozone levels react with antioxidants and oxidise lipids without affecting stratum corneum enzymes.

In conclusion, effects of ozone on antioxidant levels and lipid peroxidation were observed in the murine and human skin at a concentration of 1 ppm in air after exposure of up to 2 hours. This leads to the assumption that prolonged exposure to high concentrations of ozone can cause irreversible and corrosive damage to the skin. This is further supported by its physicochemical properties as a strong oxidising agent as well as by a large amount of inhalation studies showing that acute exposure to ozone at 1-2 ppm already causes lung injury in animals in vivo. Based on this argumentation, it is concluded that highly-concentrated ozone is corrosive to tissues of living animals and it is not ethical to perform additional, in vivo experiments for classification of the pure substance.

Five experimental studies evaluating the potential of ozone to trigger eye irritation are available. As none of these studies provides sufficient information for risk assessment, they are only provided as supporting information. Furthermore, according to Annex VII column 2, it is not necessary to evaluate the eye irritation potential of a substance classified as corrosive.

Key value for chemical safety assessment

Skin irritation / corrosion

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (corrosive)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Additional information

No studies, in which internationally accepted guidelines (e.g. from OECD, EPA, etc.) were followed for evaluation of the skin irritating/corrosive properties of pure ozone, were found in the public domain or in the archives of the applicants.

 

Skin irritation

Five research studies, in which the susceptibility of the skin to low ppm levels of ozone was studied, were retrieved from the scientific literature. The study by Thiele et al. (1997a) evaluated whether acute 2 -hour in vivo exposure to high level of ozone (10 ppm) induces oxidative stress in the skin in hairless mice as measured by antioxidant depletion (vitamins C and E) and lipid peroxidation (malondialdehyde) and the localisation of this damage among the major skin layers. The study provides supporting information that with acute exposure at high levels, ozone reacts with antioxidants and oxidises lipids. In particular, the outermost skin layers were significantly depleted of vitamins E and C, and there was a significant increase in lipid peroxidation as reflected with the high measured levels of malondialdehyde. Next, the study by Thiele et al. (1997b) evaluated whether acute 2-hour in vivo exposure to increasing concentrations of ozone induces dose-related oxidative stress [as measured by antioxidant depletion (vitamins E) and lipid peroxidation (malondialdehyde)] in the stratum corneum of hairless mice and to examine the extent of this oxidative stress after exposure to ozone for multiple days. This study provides supporting information that with acute exposure, ozone reacts in a dose-related manner with antioxidants and lipids in the stratum corneum. Repeated exposure (2 hours/day for 6 days) to 1 ppm of ozone caused cumulative oxidative stress on murine skin. A later study by Thiele et al. (2003) evaluated skin barrier integrity in hairless mice following repeated exposure to ozone. Temperature-controlled trans-epidermal water loss (TEWL) was measured as an indicator for skin barrier integrity. The study demonstrated that the repeated exposure (twice daily for 2 hours/day for 7 days) to 2 ppm ozone did not affect skin integrity since no change in TEWL was observed in ozone-exposed mice. In the study by Weber et al. (1999), it was shown that exposure of hairless mice for 2 hours to 1 and 10 ppm ozone resulted in significant depletion of vitamin C, glutathione and uric acid in the stratum corneum. All three antioxidants were unaffected at exposure to 0.8 ppm ozone. This study does not report visual inspection for skin irritation. Another research study by He et al. (2006) studied the effects of 2-hour exposure to 0.8 ppm ozone on the levels of vitamin E, lipid peroxide, cutaneous bacteria and several key enzymes from the stratum corneum taken from human forearm skin samples. Visual observation and scoring revealed no skin irritation 30 minutes after ozone exposure. This study further provides supporting information that acute high ambient ozone levels react with antioxidants and oxidise lipids without affecting stratum corneum enzymes

 

In conclusion, effects of ozone on antioxidant levels and lipid peroxidation were observed in the murine and human skin at a concentration of 1 ppm in air after exposure of up to 2 hours. This leads to the assumption that prolonged exposure to high concentrations of ozone can cause irreversible and corrosive damage to the skin. This is further supported by its physicochemical properties as a strong oxidising agent as well as by a large amount of inhalation studies showing that acute exposure to ozone at 1-2 ppm already causes lung injury in animals in vivo. Based on this argumentation, it is concluded that highly-concentrated ozone is corrosive to tissues of living animals and it is not ethical to perform additional, in vivo experiments for classification of the pure substance.

 

Eye irritation

According to Annex VII column 2 of the REACH regulation, testing for eye irritation would not be carried out on substances known or predicted to be corrosive to skin. Such substances are automatically considered to be severely damaging to the eye and are classified but not labelled for serious eye damage in addition to skin corrosion. Nevertheless, five research studies, in which the susceptibility of the eye to low ppm levels of ozone was studied, were retrieved from the scientific literature and included in the dossier as supporting information as none of them provides sufficient information for risk assessment according to CLP criteria.

 

Hine et al. (1960) and Mettier et al. (1960) exposed rabbits to ozone either once at 1.9-2.8 ppm for 4 h or at 1.0 ppm for 4 h daily for 25 days. No effects of ozone treatment on the rabbit corneas were observed by ophthalmologic examination. Also, no effects of eyes from human subjects exposed for 6 minutes to 2 ppm ozone were observed by ophthalmoscopic observations. However, no data were given on the source and the monitoring of the concentration of the ozone. Therefore, the findings in this study need to be taken with caution, particularly if more recent studies have reported findings otherwise. Lee et al. (2013) exposed mice to 0.5 and 2 ppm ozone either once or 3 h daily for 2 wks. Effects on the cornea after 1- or 2-week exposure to ozone as well as 2 weeks after exposure (as recovery) were studied by means of fluorescein staining, interleukin and interferon determination in tears, tear production and Goblet cell counts. In addition, cultured human conjunctival cells were exposed to 2 ppm ozone for 0.5, 1, 3, 5 and 8 h. Cytotoxicity and expression of inflammatory proteins were studied by means of microscopy, a luciferase assay, Western blot analysis and RT-PCR. The in vivo test revealed loss of cornea integrity at both exposure levels, and no indications of regeneration were observed after the 2 week recovery period. Mucin secreting cells were decreased, and production of inflammatory cytokines was increased. In vitro changes, which were indicative of an inflammatory response, were observed. In another study on health effects on welders in a welding shop, complaints of eye irritation among workers exposed to ozone in the range from 0.8-1.7 ppm were reported (Challen et al., 1958). Klenø and Wolkoff (2004) exposed human eyes in vivo to 71 ppb of ozone for 20 minutes and measured blink frequency as a measure of trigeminal stimulation of the eye. Blink frequency during exposure was compared with blink frequencies before and after exposure. No effects of the treatment on blink frequency were observed. However, four out of eight subjects experienced some eye irritation at this exposure. In controlled human exposure studies (summarised in IUCLID section 8.12) that involved exposures to 0.1-0.4 ppm ozone for 2-8 hours, no burning sensation and/or irritation of the eyes were reported.

 

In summary, biochemical and morphological changes in the eyes of mice were already observed following a repeated exposure to 0.5 or 2 ppm of ozone with no indications of regeneration after the recovery period of 2 weeks. Welders experienced eye irritations exposed to similar levels, but these levels are approximately 10-fold or morehigher than atmospheric ozone concentrations. At ozone concentrations frequently reported in urban areas, eye blink frequency revealed no irritation, and from controlled human exposure studies, exposure to 0.1-0.4 ppm ozone for 2 -8 hours also showed no burning sensation and/or irritation of the eyes.

Justification for classification or non-classification

Effects of ozone on antioxidant levels and lipid peroxidation were observed in the murine and human skin at a concentration of 1 ppm in air after exposure of up to 2 hours. This leads to the assumption that prolonged exposure to high concentrations of ozone can cause irreversible and corrosive damage to the skin. This is further supported by its physicochemical properties as a strong oxidising agent as well as by a large amount of inhalation studies showing that acute exposure to ozone at 1-2 ppm already causes lung injury in animals in vivo. Based on this argumentation, it is concluded that highly-concentrated ozone is corrosive to tissues of living animals. Based on unpublished historic data from accidental exposure of workers it can be concluded that high concentrations of ozone do not lead to corrosive effects within the first 3 minutes. Therefore, classification as Skin Corr. 1B is considered justified.