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The SCCP opinion states:
3.3 Toxicological evaluation
3.3.1.Acute toxicity
3.3.1.1. Acute oral toxicity
* Oral LD50 - values for rats vary between 600 and 1617 mg/kg bw (Y.Li, unpublished; Ito et al., 1976).
* A 16-month-old boy (body weight 11.6 kg) was found playing with an empty bottle that had contained about 230 g of 3% hydrogen peroxide solution. The container had a cracked lid that allowed the contents to be sucked. White foam emerged from the child’s mouth and nose. He then walked to bed and was found dead 10 hours later. In a post-mortem examination there was frothy blood in the right ventricle of the heart and the portal venous system. The gastric mucosa was red and the brain oedematous. Histopathological examination showed oedema in the lungs, and diffuse interstitial emphysema was evident. Gas emboli are found within the pulmonary vasculature and gastric and intestinal lymphatics. Clear vacuoles were also found within the walls of the gastrointestinal tract, in the spleen, kidney and myocardium (Cina et al., 1994). The maximum estimated dose of hydrogen peroxide ingested was 7 g, (600 mg/kg bw).
* An uncommon route of absorption from a cavity presumably lined by well-vascularized granulomatous tissue involved an obese 54-year-old male who underwent irrigation of an infected and fistulous herniorrhaphy wound with 5 x 20 ml volume of 3 % hydrogen peroxide. Not all irrigating volume seemed to have drained from the wound. On the fifth irrigation, the patient suddenly lost consciousness, showed cardiac shock and fell to coma which lasted for 15 min. There was no indication of red cell damage. ECG showed signs of transient myocardial ischaemia. The patient made a full recovery within 3 days. The authors attributed this occurrence to widespread embolization of oxygen microbubbles, especially to the cerebral and coronary arteries (Bassan et al., 1982). If it is presumed that as much as one half of the volume of the irrigating solution was absorbed, the hydrogen peroxide dose would have been 1.5 g implying for an obese person (assumed weight of 100 kg) about 15 mg/kg bw.
* Oxygen embolism has been reported in several infants following intestinal irrigation with hydrogen peroxide to remove meconium (Danis et al., 1967; Shaw et al., 1967). In one case a 36-hour old infant died following use of 1% hydrogen peroxide to remove inspissated meconium from the bowel due to meconium ileus (Shaw et al., 1967).
Tooth whiteners containing 10-22% carbamide peroxide:
* Oral LD50 - values for rats reported >5.000 mg/kg bw (Rope [Report], 1993; Huang
[Report], 1996; Adam-Rodwell et al., 1994; Cherry et al., 1993). (It appears that LD50
studies with rats for doses less than 5.000 mg/kg bw has not been performed)
* Oral LD50 - values for mice vary between 87.2-143.8 mg/kg bw (Woolverton et al., 1993).
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The SCCP opinion states:
3.3.1.2. Acute dermal toxicity
* Dermal LD50 -values for rats vary between 700 and 7500 mg/kg bw (FDA, 1983).
* Dermal LD50 –values in rabbits about 630 mg/kg bw (FDA, 1983).
3.3.1.3. Acute inhalation toxicity
/
3.3.1.4. Summary / Comment on Acute toxicity
The oral and dermal LD50 of hydrogen peroxide in rats is higher than 600 mg/kg bw. The dermal LD50 in rabbits is 630 mg/kg bw.
A 16-month-old boy (body weight 11.6 kg) died after ingestion of about 600 mg/kg bw.
3.3.2.Irritation and corrosivity
3.3.2.1. Skin irritation
* Skin irritation tests in rabbits with concentration of hydrogen peroxide of 3-8% were non- irritating to intact and abraded skin following exposure for 24 hours under occlusive dressing (cited in ECETOC, 1996). Irritation was slight following 4 hour exposure to 10% hydrogen peroxide and mild with 35% hydrogen peroxide. Desquamation occurred in 2 of 6 animals at day 14 with the latter concentration (Aguinaldo et al. [Abstract], 1992).
Tooth whiteners containing 10-22% carbamide peroxide:
* Primary irritation of the skin of rabbits was not found with tooth whitener (Rope [Report], 1993).
3.3.2.2. Eye irritation
* Eye irritation studies with rabbits indicate that a 5% hydrogen peroxide solution is non- irritant to mildly-irritant (Weiner et al. [Abstract], 1990).
* Several drops of a 2-5% solution induced much clouding of the cornea and inflammation of the conjunctiva of rabbit eyes. A 1% solution applied repeatedly caused conjunctival hyperaemia and slight corneal haze, followed by recovery (Koster, 1921 as quoted by Grant, 1986).
* Testing of eye irritancy for hydrogen peroxide with the Draize method indicated that 5% solution was slightly irritating (FMC, 1987a), 8% solution was moderately irritating (EU classification irritating) (FMC, 1987b), and 10% solution was highly irritating (EU classification risk of serious damage to eyes) (FMC, 1985).
* A woman who had inadvertently stored a contact lens in a 3% hydrogen peroxide disinfectant solution experienced hyperaemia, tearing, and eyelid spasm (Knoph, 1984).
* In 10 human volunteers, the threshold of detection for irritation was about 0.1% when hydrogen peroxide was administered as drops directly to the eye (McNally, 1990).
* When a hydrogen peroxide solution was administered to the eye of human volunteers via soaking contact lenses, the threshold of detection for hydrogen peroxide irritation was less than 0.03% (McNally, 1990).
3.3.2.3. Mucous membrane irritation
* 1 or 1.2% hydrogen peroxide applied to the gingivae or tongues of anaesthetised dogs by continuous drip caused oedema, followed by destruction and sloughing of the cornified epithelial layer of the gingivae (Martin et al., 1968, Dorman and Bishop, 1970).
Tooth whiteners containing 10-22% carbamide peroxide:
* No evidence of oral mucosal irritation after applying tooth whiteners containing 10% or
22% carbamide peroxide for up to 6 week in experiments with rats, hamsters and rabbits has been reported (Rope [Report], 1993; Huang [Report], 1996; Adam-Rodwell et al.,
1994; Li et al. [Abstract], 1996; Webb [Report], 1996).
* Stomach gavage of 15 and 50 mg/kg bw carbamide peroxide or 150 and 500 mg/kg bw of a tooth whitener agent (Opalescence) containing 10% carbamide peroxide produced ulceration of gastric mucosa. No ulceration was observed with 5 mg/kg bw carbamide peroxide. The lesions were clearly visible after 1 hour and seemed to be healing after 24 hours. The ulcerations of the gastric mucosa were more pronounced after exposure to the tooth-bleaching agents than those observed after a comparable dose of carbamide peroxide. The authors point out that this may be attributed to the hydrophobic gel and the content of a carbopol (which increases the tissue adherence and retards the release of oxygen) in the bleaching agent (Dahl and Becher, 1995).
* Stomach gavage of doses up to 2,000 mg/kg bw of tooth whiteners containing 10% carbamide peroxide or 70 mg hydrogen peroxide was given weekdays for 15 weeks or 6 months to Chinese hamsters. Cyclophosphamide and water served as control substances.
(Concentration and results with cyclophosphamide not stated). Histopathological findings of the gastroduodenal tissue were comparable among the groups (Li et al. [Abstract],1993).
3.3.2.4.Summary / Comment on Irritation and Corrosivity
4 hour exposure to 10% hydrogen peroxide caused slight skin irritation in rabbits. A 5% solution of hydrogen peroxide was slightly irritating to the eye while a 10% solution was highly irritating. The threshold of detection for irritation was about 0.1% when hydrogen peroxide was administered as drops directly to the human eye.
Stomach gavage of 15 mg/kg bw of carbamide peroxide (5.4 mg/kg bw of hydrogen peroxide) produced ulceration of gastric mucosa in rats observed after 1 hour; the lesions appeared to be healing after 24 hours. No effects were observed with 5 mg/kg bw of carbamide peroxide (1.8 mg/kg bw of hydrogen peroxide) (Dahl and Becher, 1995).
3.3.3.Skin sensitisation
* Ten guinea pigs were exposed to 3 or 6% hydrogen peroxide on intact or abraded skin and by intradermal injections of 0.1 ml of test solution in saline. Test solutions were re-applied
9 times over a 2 week period prior to a challenge to evaluate sensitisation. The final reactions did not indicate induction of skin sensitization with either solution (DuPont [Report], 1953).
* A case report observed skin sensitisation reaction from two women who had been exposed to hydrogen peroxide as an ingredient in commercial hair dyes. Both women tested positively to 3% hydrogen peroxide and numerous other ingredients in the hair dyes (Aguire et al., 1994).
* In a study of 156 hairdressers patch tested with the hairdressers series of chemicals were all negative to hydrogen peroxide (3%). The Dermatological Department at the Finnish Institute of Occupational Health has since 1985 tested dermatitis patients having had exposure to hairdressing chemicals with a series of test substances containing 3% hydrogen peroxide in water. 130 patients have been tested with no allergic reactions. One patient exhibited an irritant reaction. The Finnish Register of Occupational Diseases which was searched from 1975 through 1997 did not contain any cases of allergic dermatosis caused by hydrogen peroxide. The Dermatology Department of the University Central Hospital in Turku, Finland, patch tested 59 patients with 3% hydrogen peroxide during 1995-96. No positive reactions were found (Kanerva et al., 1998).
3.3.3.1.Summary / Comment on Sensitisation
Hydrogen peroxide is not considered to cause skin sensitisation.
3.3.4.Dermal / percutaneous absorption
After the application of 5-30% solutions of hydrogen peroxide on rat skin in vivo, some H2O2 could be localised in the excised epidermis within a few minutes. By contrast, with human cadaver skin in vitro H2O2 was detectable in the dermis only after the application of high H2O2 concentrations for several hours, or after pretreatment with hydroxylamine (inhibitor of catalase). Based on histochemical analysis, H2O2 was not metabolised in the epidermis, and the passage was transepidermal, avoiding the “preformed pathways” of skin appendages. The localisation of dermal emphysema, caused by liberation of oxygen, correlated for the most part with the distribution of catalase activity within the tissue (ECB, 2003).
3.3.4.1. Absorption from mucous membranes
Administration of hydrogen peroxide solutions to body cavities lined by mucous membranes, (such as sublingually, intraperitoneally and rectally) resulted in increased oxygen content of the draining venous blood and, if the amounts of hydrogen peroxide were sufficiently high, formation of oxygen bubbles. Mongrel dogs were treated with colonic lavage, or the lavage of small and large bowel was performed through an enterotomy with dilute saline solutions of hydrogen peroxide. Small amounts of the more concentrated solution (1.5% or higher) produced immediate whitening of the mucosa, with prompt appearance of bubbles in the circulation. More dilute (0.75-1.25%) solutions had the same effect when left in contact with the bowel for a longer time or when introduced under greater pressure or in greater volume for a given length of bowel. Venous bubbling was never observed at concentrations less than 0.75% H2O2. In none of the animals did mesenteric thrombosis or intestinal gangrene develop. Application of 1% hydrogen peroxide to the serosal membrane caused whitening due to gas filled small vessels; higher concentrations (up to 30%) on the skin and mucous membranes (of various species) caused lasting damage when subcutaneous emphysema and disturbances of local blood circulation impaired tissue nutrition (ECB, 2003).
3.3.4.2.Summary / Comment on Absorption
Biological membranes are highly permeable to hydrogen peroxide. Thus, hydrogen peroxide is expected to be readily taken up by the cells constituting the absorption surfaces, but at the same time it is effectively metabolised, and it is uncertain to what extent the unchanged substance may enter into blood circulation. Moreover, red blood cells have an immense metabolic capacity to degrade hydrogen peroxide and will remove hydrogen peroxide that might enter blood circulation.
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The SCCP opinion states:
3.3.5.1. Repeated Dose (28 days) oral / dermal / inhalation toxicity
Inhalation
Rats
Groups of five male and female Alpk:APfSD (Wistar-derived) rats were exposed whole-body for 6 hours per day to 0 (control), 2.9, 14.6 or 33 mg/m3 hydrogen peroxide vapour for 5 days per week, for a period of 28 days. Clinical signs which demonstrated respiratory tract irritation were seen at the exposure levels of 14.6 and 33 mg/m3, but not at 2.9 mg/m3. Concentration related necrosis and inflammation of the epithelium in the anterior regions of the nasal cavity was found at the two higher levels. Mononuclear cell infiltration was seen in two females at the highest exposure concentration in the larynx. Moreover, in the lungs, one male rat in each exposure group and two female rats in the top dose group exhibited perivascular neutrophil infiltration, and there was haemorrhage in some animals at the two lower dose levels. Control animals did not exhibit changes. The nasal localisation of the primary injury by peroxide is what can be expected from a water soluble oxidant vapour. As regards pathology in the lungs, the authors of the study considered it unlikely that the effects were treatment related due to the absence of a relationship with exposure concentration and the low incidence, and hence the NOAEL of the study would be 2.9 mg/m3 (CEFIC, 2002)
3.3.5.2. Sub-chronic (90 days) oral / dermal / inhalation toxicity
Mice
Study 1
Mice drinking 0.15% hydrogen peroxide (about 150 mg/kg/day) ad libitum grew normally and developed no visible abnormalities during a 35-week test period (FDA, 1983). Necropsy results show changes in the liver, kidney and stomach and small intestine. Hydrogen peroxide solutions at >1% (> 1 g/kg/day) caused pronounced weight loss and death of mice within 2 weeks (FDA,1983).
Study 2
Mice (C57BL/6N, catalase deficient) (groups of 15/sex) received solutions of 0, 100, 300, 1000 or 3000 ppm hydrogen peroxide in distilled water for 13 weeks. Control animals were given distilled water. At term, ten males and ten females from each group were anesthetised, blood samples were collected and the animals were killed for macroscopic and histopathological examination. Five animals/sex/group continued on untreated distilled water for an additional 6-week recovery period.
Treatment period (Days 0-90): Clear treatment-related, dose-dependent effects were noted among both females and males receiving 300, 1,000 or 3,000 ppm of H2O2. Body weights were significantly reduced only in male and female animals receiving 3,000 ppm. Dose-related reductions in both food and water consumption were observed in female animals receiving 300 ppm and greater, while among the males consistent reductions were observed at the top dose level. Among females 300 ppm (103 mg/kg/day) was a LOAEL based on significant reduction in water consumption.
Recovery period (Days 91-134): The most notable effect was increased water consumption observed among males that had received 3,000 ppm, and among females that had received 300, 1,000 or 3,000 ppm.
Histopathology: Histological examinations were performed on all gross lesions, on the tongue, oesophagus, stomach, duodenum, ileum, jejunum, caecum, colon, and rectum from all animals in all groups, and on all major organs including the sex organs in the high dose and control animals.
Hydrogen peroxide related changes were observed only in the duodenum at terminal sacrifice in the 1,000 and 3,000 ppm groups of males and females, and in a single 300 ppm group male. Although the general architecture of the affected duodenum was normal, there was an increase in cross sectional diameter and a larger mucosal area with broader, more substantial villi when compared to those of control mice. The change was assessed as mucosal hyperplasia because of the increase in mucosal thickness and size of the villi. Mucosal hyperplasia was not found in 100 ppm group mice, neither among controls.
Mortality: There were no treatment-related deaths. One male mouse died in the control group (the cause of death was undetermined), and one male mouse in the 3,000 ppm group died on study day 43 (no histopathological findings). After the recovery period no hyperplasia was observed in any dose group.
Conclusion: NOAEL was 100 ppm (26 and 37 mg/kg/day) for males and females, respectively, based on dose-related reductions in food and water consumption, and on the observation of duodenal mucosal hyperplasia (Weiner et al., 1998).
Rats
Study 1
When Wistar rats were administered 5% hydrogen peroxide by oral gastric tube 6 days weekly for 90 days with a dose range 56.2 to 506 mg/kg bw/day, the dose of 506 mg/kg suppressed bodyweight gain, decreased food consumption, and caused changes in haematology, blood chemistry, and organ weights. Principal organ affected was gastric mucosa, and the effect was local. The no-observed-effect-level (NOEL) of hydrogen peroxide was 56.2 mg/kg/day (Ito et al., 1976).
Study 2
In another Wistar rat study (Kawasaki et al., 1969) the rats were administered a 0.06 to 0.6% hydrogen peroxide by gavage for 100 days with a dose range 6 to 60 mg/kg bw/day. The top dose was associated with effects: a significant reduction of the body weight gain after day 20 of administration, a slightly higher spleen weight on day 40 (but not at termination on day 100), a decreased haematocrit and plasma proteins on day 100. Plasma catalase was significantly decreased at the termination on day 100 in the 30 and 60 mg/kg bw/day dose groups. Thus, NOAEL was 20 mg/kg bw/day.
Study 3
Groups of 10 male and female F344 rats were given 0, 0.15, 0.3, 0.6, 1.2, or 2.4% hydrogen peroxide in drinking water for 10 weeks (Takayama, 1980). Prominent weight losses and nasal bleeding were noted in the rats on the 2.4% solution starting immediately after initiation of the treatment. Also in the 1.2 and 0.6% dose groups, weight losses were noted from an early stage of hydrogen peroxide treatment. Regarding body weight gain, a gain rate of 66.1% was achieved in the male controls, whereas a maximum gain of 53.3% was achieved in the hydrogen peroxide treated groups (0.6%), and a 45.9% weight loss occurred in males on 2.4% hydrogen peroxide. A gain rate of 37.2% was found in female controls, whereas a maximum gain of 29.7% was achieved in the low dose (0.15%) hydrogen peroxide group, and a weight loss of 30.4% in the top dose group. Nine of the males on the 2.4% solution and all rats at other dose levels survived the 10-week treatment. As in the male groups, nine of the 10 females at the top dose level and all animals at the other dose levels survived the treatment. Histopathology was performed on 5 rats in each group. Pathological findings were made only at the top dose level: all males and females exhibited multiple gastric erosions and ulcer, 2 males showed atrophy of testis (in the whole group testis weights were reduced by 60% compared to controls), one rat showed congestion of the liver (died at week 7). The losses in weight of tissues other than the brain in the top dose males roughly corresponded to the body weight loss, the same applied for females. In view of the apparent effect on the weight gain even at the lowest dose level, no NOAEL can be determined.
Conclusion
A LOAEL can be calculated to 75 and 86 mg/kg bw/day for male and female rats, respectively.
3.3.5.3.Chronic (> 12 months) toxicity
See section 3.3.7 Carcinogenicity
3.3.5.4.Summary / Comment on Repeated dose toxicity
Comment
In a 90 day study in mice (Mice study 2) with hydrogen peroxide in the drinking water, a NOAEL of 100 ppm was found based on dose-related reductions in food and water consumption, and on the observation of duodenal mucosal hyperplasia. This corresponds to 26 and 37 mg/kg bw/day for males and females, respectively. In a 100 days rat gavage study (Rat study 2), a NOAEL of 20 mg/kg bw/day was found based a significantly reduced plasma catalase level at higher dose levels.
A NOAEL of 20 mg/kg bw/day may be used for calculation of MOS [Margin Of Safety]
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The SCCP opinion states:
3.3.6.Mutagenicity / Genotoxicity
The in vitro and in vivo genotoxic potential of H2O2 is summarised in Table 3.1 and 3.2, respectively.
Table 3.1: Summary of in vitro genotoxicity of hydrogen peroxide (IARC, 1999)
Table 3.2: Summary of in vivo genotoxicity of hydrogen peroxide (ECB, 2003)
Table 3.2 (contd)
aThe full references of the articles in Tables 3.1 and 3.2 are given separately at the end of the Opinion
In addition to the studies reported above it has been found that hydrogen peroxide in concentrations of 0.2 µg/ml induces cell transformation in the Syrian hamster embryo assay (Mikalsen et al., 1990). Hydrogen peroxide enhanced N-methyl-N-nitrosourea (MNU)-initiated transformation of MYP3 cells, an anchorage-dependent non-tumorgenic rat bladder epithelial cell line. Moreover, hydrogen peroxide treatment alone also caused transformation. The transformants induced by MNU plus hydrogen peroxide or hydrogen peroxide alone formed high-grade transitional cell carcinomas when injected into nude mice (Okamato et al., 1996). Hydrogen peroxide inhibited gap junction intercellular communication (GJIC) in WB-F-344 rat liver epithelial cells with an I50 of 6.8 µg/ml. The results indicated that the effects were not caused by free radical damage (Upham et al., 1997). In other systems it has been found that hydrogen peroxide enhances GJIC (Mikalsen and Sanner, 1994).
Tooth whiteners
The genotoxicity of tooth whiteners has been investigated in a number of studies. Two studies (Adam-Rodwell et al., 1994; Lee [Report], 1996) found that tooth whiteners containing 10% carbamide peroxide were not mutagenic in the Salmonella test. Other studies showed a dose response effect of tooth whiteners containing 10% carbamide peroxide in TA102 when tested without S9 (Li et al. [Abstract], 1992; Li [Report], 1997). In the test with S9, the tooth whiteners were not mutagenic. When comparing data obtained from hydrogen peroxide and carbamide peroxide examined in the same test, the observed effect of tooth whiteners appears to be associated with their peroxide contents (Li et al. [Abstract], 1992; Li [Report], 1997). The effect of four bleaching agents containing hydrogen peroxide or carbamide peroxide was studied in different E. coli strains with various capabilities to repair damages to DNA. The bleaching agents tested decreased the survival fractions of all strains studied and the effect was greatest on the strains with the lowest ability to repair DNA damage. The authors conclude that the results on dental bleaching agents generate biological effect like the ionising radiations and that their use must be strictly controlled by a dentist in order to prevent any contact with gingival and mucous tissues (Zouain-Ferreira et al., 2002).
Ribeiro and coworkers (2005, 2006) have recently assessed the genotoxicity of six commercial dental bleaching agents by the single cell gel (comet) assay in vitro with mouse lymphoma cells and Chinese hamster ovary (CHO) cells. All compounds tested induced DNA damage as depicted by the mean tail moment. The strongest effect was observed with the highest dose of hydrogen peroxide (35%). The authors concluded that dental bleaching agents may be a factor that increases the level of DNA damage.
Whitening gel containing hydrocarbon-oxo-borate complex were compared with commercial hydrogen peroxide and carbamide peroxide products. The effects of human epithelial cell line for induction of DNA damage and subsequent induction of apoptosis and necrosis have been studied. The study was used in MCF-7 (human breast cancer cells). The result show that the two hydrogen peroxide and the one carbamide peroxide based products induce significant DNA breakdown in MCF-7 cells while the hydrocarbon-oxo-borate complex showed much less DNA breakdown even at the highest concentration. While the hydrogen peroxide and carbamide peroxide bleaching agents induced massive necrosis at both 1 mg/ml and 10 mg/ml and no induction of apoptosis, the borate gel induce physiological cell death (apoptosis), both at 1 mg/ml and 10 mg/ml, while virtually no necrosis was found (Li and Ramaekers, 2004).
Several in vivo studies on peroxide containing tooth whiteners detected no genotoxicity. No increased frequency of micronuclei was observed in bone marrow cells of mice that were gavage-fed with two solutions containing 10% carbamide peroxide (Woolveton et al., 1993). Three tooth whiteners containing 10% carbamide peroxide did not increase the SCE frequency in bone marrow cells of Chinese hamsters and mice after the animals received doses up to 10 g/kg (Li et al. [Abstract], 1992, 1993; Lee [Report], 1996). Also using the SCE assay, a tooth whitener paste containing 10% carbamide peroxide was found to be non-genotoxic when administered to rats at doses ranging from 0.1 to 1.0 g/kg for 5 days (Adam-Rodwell et al., 1994). A long term study showed that oral administration of tooth whiteners of 10% carbamide peroxide up to 2 g/kg daily on week days for 3 or 6 months did not affect the SCE frequency of bone marrow cells of Chinese hamsters (Li et al.[Abstract], 1993).
Conclusion of the European Chemicals Bureau on the mutagenicity of hydrogen peroxide (ECB, 2003)
Hydrogen peroxide is a mutagen and genotoxicant in a variety of in vitro test systems. The responses observed were modified by the presence of degrading enzymes (catalase), the extent of formation of hydroxyl radicals by Fenton reaction, and the cells repair abilities.
Hydrogen peroxide has been studied for possible in vivo genotoxicity. Studies employing modern methodologies have explored DNA repair in liver cells of rats administered hydrogen peroxide by intravenous infusion for 30 minutes (CEFIC, 1997), as well as micronucleus formation in mice in the context of a 2-week drinking water exposure (Du Pont, 1995), or after a single intraperitoneal injection (CEFIC, 1995), all with a negative outcome. Intravenous administration of hydrogen peroxide in the in vivo-in vitro unscheduled DNA synthesis study ensured that the substance had a fair chance to reach the target (liver) cells, although the duration of exposure was limited (CEFIC, 1997). In the micronucleus study by oral drinking water exposure (Du Pont, 1995), the systemic fate of hydrogen peroxide was uncertain, and there was no decrease in the ratio of polychromatic/normochromatic erythrocytes in the bone marrow. In the other micronucleus study (CEFIC, 1995), a single intraperitoneal injection of a large dose of hydrogen peroxide somehow affected the bone marrow (because the PE/NE decreased), but the absence of micronucleus formation must be viewed with caution because of the presumably very short lifetime of hydrogen peroxide. With a view to exploring target tissue in vivo genotoxicity and mutagenicity as a pre-screen for carcinogenicity, hydrogen peroxide 0.2-3.2% solutions in ethanol were applied to the skin of Sencar mice twice weekly for 4 weeks (Society for Plastic Industry, 1997). There was no indication of induced DNA damage (increased 8-OH-dG), c-Ha-ras mutations, epidermal hyperplasia and dermal cellularity changes. Thus, at low concentrations, and with a low application frequency, hydrogen peroxide did not induce local mutagenicity in this tissue model.
In conclusion, the available studies are not in support of a significant genotoxicity/mutagenicity for hydrogen peroxide under in vivo conditions. A wider database of genotoxicity and mutagenicity observations on other relevant target tissues in direct contact with hydrogen peroxide is, however, desirable. Mechanistic studies suggest that cells are adapted to repair DNA damage caused by oxidants; on the other hand there is some evidence that hydrogen peroxide may inhibit the repair of DNA lesions inflicted by other types of reactive chemicals (Churg et al., 1995, Pero et al., 1990, Hu et al., 1995).
According to the principles followed in the EU, hydrogen peroxide is not classified as a mutagen.
3.3.6.1. Comment on the Conclusions of the European Chemicals Bureau
The SCCP endorses the conclusions of the European Chemicals Bureau.
3.3.7. Carcinogenity
Mice
Oral
C57BL/6J mice, groups of 50 males and 50 females (eight weeks old) were given 0 (control), 0.1, and 0.4% hydrogen peroxide in drinking water for 100 weeks. The bodyweight of the hydrogen peroxide treated groups were comparable to those of control mice except for a slight decrease in the bodyweight of females of the 0.4% group at 15 months of age. Survival among control mice (54%) was lower than for mice treated with hydrogen peroxide (63% for high dose and 61% for low dose). An increased frequency of tumours in the duodenum was found (Table 3.2) (Ito et al., 1981).
When the data for male and female mice were combined (Ito et al., 1981), there was a statistically significant increase in the incidence of duodenal carcinomas, but when treated separately and analysed statistically with Fisher’s Exact Test, there was no significant difference between dosage groups. Ito et al. (1981) reported invasion of the duodenal carcinomas into the muscular layer and small vessels, but no metastatic tumours were evident. No treatment-related tumours were noted elsewhere. The latency of tumour induction was decreased in the treated mice, the first lesion occurring at about 42 weeks in mice treated with 0.4% H2O2. The decreased latency was based on animals that died and not those from interim kills. The authors suggested that the neoplastic nodules developed mainly in the duodenum because H2O2 is unstable under alkaline conditions.
A group of 138 male and female C57BL/6N mice was treated with 0.4% hydrogen peroxide in the drinking water. Groups of 5-17 mice were killed sequentially at 30-day intervals up to 210 days and then every 60, 70 or 90 days up to 630 days; 29 mice were killed on day 700, when the experiment was terminated. Gastric erosions appeared at the first kill (30 days) and were present consistently at each subsequent kill. “Nodules” (hyperplastic lesions, adenomas and carcinomas) were found in both duodenum and stomach from 90 days until the end of the experiment, but not on days 210 and 360 in the stomach. The lesions did not appear to increase in frequency, but atypical hyperplasia appeared late in the experiment, and 5% of the animals developed duodenal adenocarcinoma. No such lesion was observed in controls. The reversibility of the lesions was investigated in groups of mice treated with 0.4% hydrogen peroxide for 120, 140, 150 or 180 days after a treatment-free period of 10-30 days. The stomach lesions regressed completely, irrespective of length of treatment, but some of the duodenal lesions persisted. Groups of 22 DBA/2N, 39 BALBcAnN and C57BL/6N mice of both sexes were given 0.4% hydrogen peroxide in drinking water. The mice were examined sequentially from 90 to 210 days of treatment for strain differences in the development of gastric and duodenal “nodules” (hyperplastic lesions, adenomas and carcinomas). The incidences of gastric nodules were 2/22 (9%), 1/39 (3%) and 12/34 (35%), and those of duodenal nodules were 14/22 (64%), 7/39 (18%) and 22/34 (65%) in DBA, BALB/c and C57BL mice, respectively. The duodenal nodules appeared at 90 days in all three strains (Ito et al., 1982).
Groups of 18-24 female C3H/HeN, B6C3F1 and C3H/Cs mice with different levels of catalase activity in the duodenal mucosa were given 0.4% hydrogen peroxide in drinking-water for 6 or 7 months. The incidence of duodenal “nodules” (hyperplastic lesions, adenomas and carcinomas) is shown in Table 3.3 (Ito et al., 1984). (The IARC Group noted that the pathology of the tumours was not well documented.)
Rats
Oral
Hydrogen peroxide was administered to Fischer 344 rats in drinking water at concentrations of 0%, 0.3% or 0.6% for 78 weeks followed by a 6-month recovery phase. Survival was similar to that of the controls (41/50), except for male rats in the 0.3% group (approximately 30% mortality; 36/50 alive at 97 weeks). Tumours of the testes, mammary gland and skin were observed in rats that died during the study; there were no differences in tumour incidence between control and treated rats. After 45 weeks of administration, body weight was decreased by about 6% in male and female rats in the 0.3% group and 10% in the 0.6% group. Nasal bleeding was observed in the treated groups; the significance of this is uncertain. At the end of the study (104 weeks), all surviving animals were killed. No significant differences were observed between treated rats and controls relative to the incidence and types of tumours. The authors concluded that, under the conditions of this study, hydrogen peroxide was not carcinogenic to Fischer 344 rats. Because this study was not published in detail, its quality cannot be assessed. Furthermore, no account was taken of other measurements made during the study, and a full characterisation of the pathological changes was not given (Ishikawa and Takayama [Abstract], 1984).
In other studies, forestomach papillomas were observed in rats exposed to hydrogen peroxide in drinking water (1%) (see Takahashi et al., 1986 [below]).
Hydrogen peroxide in initiation – promotion experiments
Mice
Skin painting
Groups of 60 female Sencar mice, aged 7 to 9 weeks, were used to test the tumour-promoting (A), tumour-initiating (B) and complete carcinogenic (C) activity of hydrogen peroxide on the skin. Mice in experiment (A) received a single topical application of 10 nmol DMBA in 0.2 ml acetone, followed one week later by applications of a 30% solution of hydrogen peroxide diluted 1:1 (15%) (once and twice weekly), 1:2 (10%) or 1:5 (5%) in 0.2 ml acetone twice weekly for 25 weeks. Controls received acetone alone. The proportions of mice with papillomas at 25 weeks in experiment A (tumour-promoting) were 0/60 (0%) (controls), 3/58 (5%; 5%HP), 5/59 (9%; 10%HP), 6/59 (10%; 15%HP once weekly) and 6/60 (10%; 15%HP twice weekly), respectively. Mice in experiment B (tumour-initiating) received a single topical application of hydrogen peroxide diluted 1:1 (15%) in 0.2 ml acetone, or acetone alone (controls), followed one week later by twice-weekly applications of 2 µg 12-O-tetradecanoylphorbol 13-acetate (TPA) in acetone for 25 weeks. Papillomas were found after 25 weeks in 3/56 (5%) and 6/58 (10%) control and hydrogen peroxide-treated animals, respectively. Mice in experiment C (complete carcinogen) received twice-weekly topical applications of hydrogen peroxide diluted 1:1 (15%) in 0.2 ml acetone for 25 week; 3/57 (5%) had papillomas at that time. No squamous-cell carcinoma was found when these animals were observed up to 50 weeks (Klein-Szanto and Slaga, 1982) (The IARC Working Group noted the absence of a DMBA-treated control group for the promotion experiment and the short duration of the experiment for complete carcinogenicity evaluation).
Comment
Mahony et al (2006) note that the authors of the report had stated that “hydrogen peroxide has extremely weak tumour promoting activity” Due to the lack of a DMBA-treated control group it is not possible to determine to potency of hydrogen peroxide as a promoter.
In similar studies, mice were treated dermally for up to 58 weeks with 3% or 5% hydrogen peroxide following initiation with DMBA (Shamberger, 1972; Bock et al., 1975; Kurokawa et al., 1984). In these studies there were no significant increases in the incidence of skin tumours, although epidermal hyperplasia was evident in most of the mice treated.
Rats
Oral
Takahashi et al. (1986) examined the potential of hydrogen peroxide to promote N-methyl-N’- nitro-N-nitrosoguanidine (MNNG) initiated gastric tumours in rats. Two groups of rats (n=30 and 21) received MNNG-treated drinking water and food supplemented with 10% sodium chloride, the water of one group being supplemented with 1% hydrogen peroxide for 7 weeks ad libitum after which the animals were maintained on normal food and tap water. A third group (n=10) was not given MNNG or a sodium chloride supplemented diet, but was administered 1% hydrogen peroxide in the drinking water.
Table 3.5. Effect on gastro-duodenal carcinogenesis induced by MNNG (Takahashi et al, 1986)
Adenocarcinomas were observed in the pyloric stomach and duodenum of the MNNG-treated rats, and “preneoplastic hyperplasia” was observed in the pylorus (Table 3.4). In rats treated with MNNG and hydrogen peroxide, there was no enhancement in the number of gastrointestinal tumours, although all treated animals exhibited forestomach papillomas; these also occurred in rats treated only with hydrogen peroxide in the drinking water. No carcinoma development was noted in the stomach or duodenum. Erosions and ulcerations also occurred in the fundic mucosa of the stomach of the hydrogen peroxide treated rats. The authors concluded that, in contrast to the study of Hirota and Yokoyama (1981, see below), no enhancement of duodenal tumours occurred, although characteristic diffuse lesions, showing fusion of the villi, were observed throughout the duodenum.
In a study (Hirota and Yokoyama, 1981) of promoting effects in intestinal carcinogenesis, groups of 3 or 8 male F344 rats were administered 1.5% H2O2 in drinking water either with or without methylazoxymethanol acetate (MAM) treatment (three i.p. injections of 25 mg/kg bw every other week) for 10 or 21 weeks; 3 control rats received water. Rats given H2O2 four weeks prior to MAM injections, during intervals between injections, and until the termination of the study showed higher incidences of duodenal (8/8, 100%) and jejunal (5/8, 63%) carcinomas when compared to rats otherwise similarly treated but not given H2O2 subsequent to MAM injections (2/8, 25% and 2/8, 25%, respectively). The three rats given H2O2 alone throughout the study period did not develop carcinomas in the studied organs; there was no group of animals receiving MAM alone. Only gross tumours of the g-i tract were reported.
The authors concluded that hydrogen peroxide had a tumour promoting effect on MAM-initiated intestinal tumours. Because of the lack of a MAM control group and details of the method, it is not possible to evaluate this study.
Hamsters
Buccal pouch painting
DMBA and/or hydrogen peroxide was painted onto the left buccal pouch of 4 groups of male Syrian golden hamsters twice weekly for 19 or 22 weeks. Animals in Group A were painted twice weekly with a 0.25% solution of DMBA in heavy mineral oil. Animals in Group B were painted twice weekly with DMBA and twice weekly (on days other than the DMBA painting) with 3% hydrogen peroxide. Group C was painted in exactly the same way as Group B animals except that the concentration of hydrogen peroxide used was 30%. Group D animals were painted twice weekly with 30% hydrogen peroxide alone. Cheek pouches from animals that had not been painted and from animals that had been painted twice weekly with only the mineral oil vehicle, served as controls. Six of 11 hamsters (55%) treated with DMBA and 3% H2O2 developed epidermoid carcinomas by 22 weeks, whereas all 5 (100%) hamsters treated with DMBA and 30% hydrogen peroxide developed epidermoid carcinomas by 22 weeks. No carcinomas were observed in hamsters treated with 30% hydrogen peroxide alone, but 3/7 (43%) of the hamsters treated with DMBA alone developed carcinomas. In all hamsters, chronic inflammation, hyperchromatic cells and dysplasia were also noted at 19 weeks. The authors concluded that longterm, twice weekly application of 3% or 30% hydrogen peroxide could induce inflammatory changes, but that pathological changes associated with preneoplastic lesions and augmentation of the oral carcinogenesis of DMBA were observed only with 30% hydrogen peroxide. The experiment demonstrated a promoting effect of hydrogen peroxide (Weitzman et al., 1986).
Marshall et al. (1996) used groups of 25 hamsters of each sex. The cheek pouches of one group were exposed to a solution containing 0.75% hydrogen peroxide together with 5% baking soda 5 times per week for 20 weeks. Another group received the 0.5% DMBA together with 5% baking soda. A third group received a commercial dentifrice containing 3% hydrogen peroxide together with 0.5% DMBA. A control group received 0.1 ml mineral oil. At the end of the treatment 0/50 (0%) of the first group developed tumours, while 40/50 (80%) of the second group developed tumours. 37/50 (74%) of the third group developed tumours.
In a second phase of the previous study, Marshall et al. (1996) applied 1.5% hydrogen peroxide in dentifrice formulation (single or dual phase) mixtures with sodium bicarbonate (7.5%) to the cheek pouches of groups of 25 male and 25 female hamsters. Another group of hamsters was exposed to a solution of 3% hydrogen peroxyde/7.5% baking soda. In each of these groups, the cheek pouches were co-treated with DMBA at concentrations of either 0.25% or 0.5%. DMBA applications were made 3 times per week while the dentifrice/solutions were administered 5 times per week. In this second phase, there was no hydrogen peroxide/baking soda exposure only group. Since the cheek pouch carcinoma incidence was close to 100% in the DMBA-only groups as well as in the DMBA/hydrogen peroxide groups, this phase of the study was not capable of detecting any potential enhancing effect of hydrogen peroxide.
Mice
Skin painting
In a skin painting experiment 30 female Swiss mice (55-69 days old) were painted once with 125µg DMBA and after 3 weeks treated 5 times weekly with 5% carbamide peroxide in water. No tumours were found when the experiments were terminated after 56 weeks of promoting stimulus. In a similar experiment with 0.1% perbenzoic acid in acetone, about 40% developed skin tumours and 10% skin cancer (Bock et al., 1975).
Hamster
Buccal pouch painting
A hamster cheek pouch study was done by Collet et al. (2001) with a 10% carbamide peroxide (~3.3% hydrogen peroxide) tooth bleaching gel. Tumours were apparent in all male hamsters (10/10; 100%) that received an oral cavity application of 0.5% DMBA three times a week for 14 weeks. No tumours were observed in hamsters treated with a combination of 0.5% DMBA twice a week and the 3.3% hydrogen peroxide gel once a week for 34 weeks or in hamsters treated with only the 3.3% hydrogen peroxide gel three times a week for 34 weeks.
Tooth whiteners
Human case report
In a press release from American Head and Neck Society a study presented at the 6th International Conference on Head and Neck Cancer is reported. Patients with primary oral cancer diagnosed at Georgetown University Medical Center between 1997 and 2003 were identified. Nineteen patients agreed to participate in the study. Three (16 percent) of patients reported a history of tooth whitener use in the past. There was no significant difference in age at diagnosis between the patients who used and did not use tooth whiteners, however the tooth whitener users tended to be younger (mean age 34.3 vs. 52.4, p = 0.11). Alcohol use and smoking history were similar in the two groups. The patients who used tooth whiteners were more likely to present with regional lymph node disease, than those who did not use tooth whiteners. All three patients presented with node positive disease as opposed to 3 of 16 (19 percent) patients without a history of tooth whitener use. The authors point out that the data do not necessarily suggest a causative relationship between the use of these products and the development of oral cancer. However, free radicals generated in the whitening process have carcinogenic potential, and therefore the use of these products in this patient population should be studied further (Burningham et al., 2004).
3.3.7.1 Discussion on carcinogenicity
Dermal exposure may be a useful model for addressing carcinogenicity in the oral cavity. The dose expressed as mg/cm2 has been established for risk assessments focused on local effects following topical application of a chemical, e.g., contact sensitisation.
Dose per unit area in dermal tumour promotion assays (assuming application to 6 cm2 area) is presented in Table 3.5 (table included in Submission III except that the experiment by Klein- Szanto and Slaga (1982) was not included in the submission and was added later by SCCP).
The doses shown refer to dermal applications in non-clinical studies evaluating tumour promotion potential. No carcinogenic effects were observed in the three first experiments. In the first hamster study it is not possible to determine if hydrogen peroxide had any promoting effect as the tumour frequency with DMBA alone was very high (80%). No tumours were found in the second hamster experiment.
In the study by Klein-Szanto and Slaga (1982) the tumour frequency was 5% in Sencar mice after initiation with the initiator DMBA followed skin painting with hydrogen peroxide for 25 weeks corresponding to about 0.47 mg/cm2/day. The peak exposure in the saliva after teeth bleaching with 5% Whitestrip was 0.1% hydrogen peroxide (Hannig et al., 2003) corresponding to about 5 µg/cm2 which is only about 100 times lower that the concentration inducing a tumour frequency of 5% in the mice skin painting study.
Mahony et al (2006) claim that 0.71 and 0.41 mg/cm2 represent a NOEL for tumour promotion in mouse skin and hamster cheek, respectively. Based on an assumed maximal human exposure of 1.5 µg/cm2, they calculated a MOS of 473 and 287 based on the mouse and hamster study, respectively. Based on the results of Klein-Szanto and Slaga (1982) as discussed above, this calculations can be questioned.
Munro et al. (2006a,b) are of the opinion that the available genetic toxicity and animal toxicology data do not indicate that hydrogen peroxide poses a carcinogenic risk to the human oral mucosa. They claim that their conclusion is supported by the results of the dosimetric exposure analyses from tooth whitening product users. Tredwin et al. (2006) and Naik et al. (2006) on the other hand point out that several carcinogenesis studies, including the hamster cheek pouch model, indicate that hydrogen peroxide might possibly act as a promoter and that urgent clinical studies are required on the genotoxic and tumour-promoting effects of hydrogen peroxide bleaching agents. They conclude that in the light of the concern regarding the possible tumour-promoting ability of H2O2 with the tobacco carcinogen DMBA, patients should be advised to avoid smoking and alcohol.
ECB (2003) note that the weak effect found in complete carcinogenesis studies in mice as well as in some promotion studies suggest promotion type of activity and possible underlying genotoxic mechanisms.
A drinking water study in mice showed that hydrogen peroxide caused duodenal hyperplasia at a high frequency and localised duodenal carcinomas at a low frequency. A subsequent study with different strains of mice showed a strong negative correlation between incidence of duodenal tumours and catalase activity in duodenal mucosa. In one study with rats a high incidence of forestomach papillomas were found after receiving 1% hydrogen peroxide in the drinking water. While humans do not have a forestomach, they do have comparable squamous epithelium tissues in the oral cavity and the upper 2-3 of the oesophagus. Thus, in principle, carcinogens targeting the forestomach squamous epithelium rodents are relevant for humans. Also, the target tissues for carcinogens may differ between experimental animals and humans and a forestomach carcinogen in rodent may target a different tissue in humans (IARC, 2003). Some tumour promotion studies indicate that hydrogen peroxide may act as a weak promoter.
Hydrogen peroxide has a weak potential to induce local carcinogenic effects. The mechanism is unclear, but a genotoxic mechanism cannot be excluded. As regard to tumour promotion, several mechanisms might be operative; direct genotoxicity, impairment of DNA repair, and chronic inflammation.
Source & ©: SCCP,
The SCCP opinion states:
3.3.8. Reproductive toxicity
Hydrogen Peroxyde
Fertility
Wales et al. (1959) gave 0.33, 1 or 3% hydrogen peroxide in drinking water to three groups of 12 male albino mice. Solutions were changed twice weekly. The mice on the high level of peroxide (3%) refused to drink and after 5 days were removed from the experiment having lost about 20% of their body weight. The remaining two groups were each divided at random into four subgroups of 3 animals. Two female mice were placed with each male of the first subgroup on day 7 and again (with two other females) on day 28 after starting hydrogen peroxide. Two subgroups of males were placed with females on day 21: the animals in one of the groups continued on hydrogen peroxide, for the other group hydrogen peroxide was replaced with tap water (ensuring no consumption of hydrogen peroxide by the females). The fourth subgroup of three male mice was killed on day 21 and the epididymal spermatozoa were examined. The drinking water of three albino rabbits was also replaced with 0.33, 1 or 3% hydrogen peroxide and the semen was examined at weekly intervals for 6 weeks. All female mice mated to treated males became pregnant within a few days and in each case healthy young were born in litters of normal size. Pregnant mice that continued to consume 1% H2O2 in water up until near term showed some delay in parturition compared to dams using tap water (the effect was, however, small and inconsistent). The concentration, morphology and motility of the mouse spermatozoa (in three mice) after 3 weeks of treatment appeared normal. There were no detectable abnormalities in the sperm of the three rabbits exposed for 6 weeks either. No firm conclusions can be drawn from this limited study which did not use any control animals, although any major deleterious effects by the treatment on reproduction seem to be excluded.
Rats
Three weanling Osborne-Mendel female rats were given 0.45% H2O2 in drinking water and maintained on it for 5 months. Thereafter they were given tap-water and mated with normal males. Six normal male litter mates were divided into two equal groups: one received 0.45% H2O2, while the other received tap water. These animals were maintained on their respective regimens for 9 months. Normal litters were produced, and thus long-term treatment with peroxide did not appear to affect the reproduction in female rats. Regarding observations made on the six male offspring that were followed for 9 months, the only noticeable effect was a difference in body weight: an average of 521 g for those on tap water against 411 g for those on H2O2 (Hankin, 1958). No firm conclusions can be drawn from this restricted study with few animals.
Male and female rats were administered hydrogen peroxide daily by gavage at doses of 1/10-1/5 LD50 (which was not specified) for 45 days. At the high dose, females showed modifications of the oestrus cycle and males reduced mobility of spermatozoa, without effects on the weight of the testicles. In a second experiment male and female rats received daily doses of 0.005, 0.05, 0.5, 5 and 50 mg hydrogen peroxide/kg bw by gavage for 6 months and were mated. Variations of the oestrous cycle in females were observed at 50 and 0.5 mg hydrogen peroxide/kg bw, but not at 5 mg/kg bw. Reduced mobility of spermatozoa in males was observed at 50 mg hydrogen peroxide/kg bw. No changes were observed in the morphology and weight of the testes. Among the high dose females, only 3/9 produced litters, compared to 7/9 in the control group. In addition, litter size and body weight gain of the offspring of the high dose females were reduced relative to those of control females (Antonova, 1974). The results of the study should be considered with caution because the information on the experiment is incomplete.
Developmental
One study which addresses developmental toxicity has been conducted with Wistar rats Moriyama et al., 1982). Aqueous solutions of hydrogen peroxide were mixed with powdered feed to 10, 2, 0.1, or 0.02% and administered to groups of 5-8 pregnant rats for one week during “the critical period of pregnancy”. The foetuses were removed on day 20 for examinations (Study A). Separate dose groups of 2-3 rats were similarly treated, but the rats were allowed to go through normal delivery, and the offspring were followed-up for about four weeks (Study B). In Study A, at the high dose level the dam body weight did not increase markedly. Food consumption was reduced to about one third as compared to the other dose groups, for which there was no difference from controls. Foetal resorptions were increased and the foetal body weight was decreased; most of the foetuses were close to death. No external malformations were found in any of the dose groups. Haemorrhaging of internal organs (eye, parietal region of the brain, cardiopulmonary region, torso) was dose dependently increased in the dose range 0.1-10% H2O2. Skeletal hypoplasias occurred dose dependently at the two highest levels. In Study B, all the neonates of the 10% treatment group died within 1 week post partum, the body weights were low and the number of live births was decreased. In the other dose groups there were no major effects on the development of neonates. There are major uncertainties about the exposure and effect mechanism which cast doubt on the relevance of the study. H2O2 concentration in feed was reported to decrease to 1/10 after 24 hours and to virtually nil by 72 hours. The authors state that “the amount of residue was determined and consumption was estimated”; however, it is not stated how frequently fresh feed was prepared. Nevertheless, it seems likely that the dams indeed ingested hydrogen peroxide, and there was not much of an increase in dam body weight at the top dose level. There was no marked difference between the groups in placental weight. The authors proposed that the observed effects on foetal development were due to the breakdown of essential nutrients in food by hydrogen peroxide.
Tooth whitener
In order to test the effect of ingested tooth whitener on early embryo development and growth, rats were intubated with 500 mg/kg whitener on day 2 of pregnancy. It was concluded that a) ingestion of tooth whitener containing 35% carbamide peroxide causes a loss of embryos sometimes between day 2 (treatment) and day 5 (collection), but that b) day 5 embryos have the same cell number both prior to and after 24 hour culture, and c) have the same ability to implant in vitro (Redmond et al. [Abstract], 1998).
3.3.8.1 Summary / comment on Reproductive toxicity No appropriate animal studies were available for a complete evaluation of reproductive and developmental toxicity. Limited studies with mice and rats exposed to hydrogen peroxide in drinking water suggested no grave disturbances on the male or female reproductive functions. The only available developmental toxicity study in Wistar rats which were fed on powdered feed mixed with hydrogen peroxide did show foetotoxic effects (Moriyama et al., 1982), but the study contains major uncertainties about the exposure and effect mechanisms. Although raising some further questions, the study cannot be used for an evaluation.
Source & ©: SCCP,
The SCCP opinion states:
3.3.9. Toxicokinetics
Hydrogen peroxide is a normal metabolite in the aerobic cells. It is produced from superoxide anion spontaneously or as a result of the activity of superoxide dismutase (SOD) (EC1.15.1.1). Superoxide radical undergoes dismutation quickly and spontaneously, but the enzymatic process occurs at a rate that is 1010-fold faster. Eukaryotic cells contain two kinds of SOD that are highly specific for superoxide (O2) as a substrate. Hydrogen peroxide occurs under most conditions at submicromolar concentrations.
O2 + e- → O2-· (superoxide anion)
O2-· + e- + 2H+ → H2O2 (spontaneous dismutation to hydrogen peroxide)
2O2-· + 2H+ → H2O2 + O2 (SOD:superoxide dismutase)
Hydrogen peroxide passes readily across biological membranes. Because it reacts slowly with organic substrates, it can diffuse considerable distances in biological systems. There are two main hydrogen peroxide metabolising enzymes, catalase and glutathione peroxidase which control the hydrogen peroxide concentration. Catalase deals with large amounts of H2O2 that may be generated in peroxisomes. Glutathione peroxidase (GSH peroxidase) metabolises H2O2 in both the cytosolic and mitochondrial compartments.
The overall decomposition reaction of hydrogen peroxide in the present of catalase is as followes:
H2O2 + H2O2 → 2H2O + O2 (catalase)
Catalases are present at a wide range of concentrations in nearly all mammalian cells. Catalases are located in subcellular compartments, mainly in peroxisomes. Soluble catalases are found in erythrocytes. The highest catalase activity is observed in cells of the duodenum, liver, spleen, kidney, blood, mucous membranes and other highly vascularised tissues.
Glutathione Glutation peroxidases decompose hydrogen peroxide through the reaction:
H2O2 + 2GSH → 2H2O+ GSSG (GSPx: glutathione peroxidase)
Glutathione peroxidase can react with both hydrogen peroxide and organic hydroperoxides. Glutathione peroxidase is more efficient at low concentrations of hydrogen peroxide compared to catalase. Glutathione peroxidase reduces hydrogen peroxide to water with formation of oxidised glutathione which is regenerated by glutathione reductase by consuming NADPH.
Relatively high peroxidase activities occur in human adrenal medulla, liver, kidney, leukocytes and saliva. In the oral cavity, salivary peroxidase and myeloperoxidase are the primary defences against bacterially derived peroxide. Salivary peroxidase activity, resulting in the conversion of hydrogen peroxide to water, is coupled with the conversion of thiocyanate to hypothiocyanate, which has bacteriostatic activity and reduces the formation of peroxide and dental plaque acid by bacteria. In the absence of salivary peroxidase and thiocyanate, the rate of production of hydrogen peroxide by bacteria in saliva is approximately 100 nmol/ml/hr and would lead to a steady-state level of 0.1 mM hydrogen peroxide in one hour (Thomas et al, 1994). In the presence of salivary peroxidase and thiocyanate, the steady-state level of peroxide was predicted to be maintained below 0.01 mM.
Significant amounts of topically applied hydrogen peroxide can penetrate the epidermis or mucous membranes followed by rapid spontaneous or enzyme-catalysed decomposition to oxygen and water in the underlying tissue. The formation of gaseous oxygen causes capillary microembolism and prevents irrigation of tissues by blood resulting in a visible, reversible bleaching of the exposed tissue area. The local spontaneous or enzymatic-catalysed breakdown prevents it to enter the general circulation and thus its systemic distribution.
The oxidative reactivity of hydrogen peroxide with biological molecules such as carbohydrates, proteins, fatty acids or nucleic acids is not pronounced in the absence of transition metals, except for a few nucleophilic reactions.
In the organism the highly reactive (and thus toxic) hydroxyl radical can be produced non-enzymatically from superoxide anion and hydrogen peroxide through catalysis by transition metal ions like Fe2+ and Cu+ (the so-called Haber-Weiss- and Fenton reactions):
metal ions
H2O2 + O2-· → OH· + OH- + O2 (Haber-Weiss reaction)
H2O2 + Fe2+/Cu+ → OH· + OH- + Fe3+/Cu2+ (Fenton reaction)
In all likelihood the “full” Haber-Weiss reaction (i.e., the reduction of H2O2 by O2-·) is as follows (showing that the Fenton reaction is representing one particular part of the Haber-Weiss reaction):
O2-· + Fe3+/Cu2+ → O2 + Fe2+/Cu+
H2O2 + Fe2+/Cu+ → OH· + OH- + Fe3+/Cu2+
Iron is normally bound and the level of free iron in the plasma is very low. The cellular iron is thus not available to mediate a Fenton reaction in vivo. Biological reducing or chelating agents, or acidic pH, may however promote the release of iron from transport and storage proteins. (ECB, 2003).
The hydroxyl radical is highly reactive and oxidises all organic chemicals, including biomolecules, when present in very close proximity to the place where the hydroxyl radical is formed. Superoxide and H2O2 are less reactive and can diffuse away from their site of formation, leading to OH· generation whenever they meet a “spare” transition metal ion. Oxygen radical formation can lead to lipid peroxidation, destruction of proteins, including enzyme inactivation, or to DNA damage.
Groups at extra risk
Genetically determined traits (acatalasaemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency) render humans more susceptible to peroxide toxicity.
Acatalasemic individuals are more susceptible to hydrogen peroxide exposure because of a hereditary disorder in their hydrogen peroxide metabolising enzymes, i.e. the blood catalase activity level is below normal (hypocatalasemia). Acatalesemia is a rare (frequency 0.2-0.4%) genetic defect occurring particularly in the Orient (Ogata, 1991). It has been found that approximately half of the Japanese acatalasemic patients developed progressive gangrene of the mouth called Takahara’s disease. This condition is characterised by small, painful ulcers in the gingival crevices and tonsillar lacunae, attributed to excess levels of hydrogen peroxide generated by various microorganisms in the mouth without normal destruction by catalase. The total number of reported patients of acatalasemia worldwide in 1989 was 107 belonging to 52 families.
Two Hungarian acatalasaemic subjects were reported (Góth, 1992). There appears to be two types of acatalasaemia. The Japanese type is the result of a splice mutation resulting in defective catalase synthesis (Góth and Páy, 1996). The Swiss type of acatalasaemia type is caused by point mutation resulting in catalase that is rapidly degraded. Swiss type acatalasaemic patients show no signs of oxidative damage (Góth and Páy, 1996).
Another group of individuals more sensitive to hydrogen peroxide exposure is persons with G6PD deficiency. G6PD deficiency is a genetic disorder of erythrocytes (over 300 variants have been identified) in which the inability of affected cells to maintain NAD(P)H levels sufficient for the reduction of oxidised glutathione results in inadequate detoxification of hydrogen peroxide through glutathione peroxidise (Gaskin et al., 2001, Tsai et al., 1998). It is estimated that about 400 million people throughout the world are deficient in G6PD. The frequency in G6PD deficiency in Europe is about 0.1%.
Industry claimed that due to the low levels of hydrogen peroxide in saliva during use of tooth whitening products and conversion of exogenous hydrogen peroxide to water and oxygen, hydrogen peroxide would not be expected to persist long enough in the body to reach G6PD deficient erythrocytes to precipitate an oxidative response.
A third group of individuals that might be more sensitive to hydrogen peroxide exposure is persons with xerostomia, or dry mouth, which occurs when the salivary glands are hypoactive. This may affect the degradation of hydrogen peroxide. However, Marshall et al. (2001) found no difference in the clearance of peroxide from the oral cavity when comparing adults with normal salivary flow and adult with diminished salivary flow (Sjorgren’s syndrome). In an industry sponsored clinical study (2000159), subjects with artificially induced xerostomia (via use of a rubber dental dam) experienced no adverse events after 10 days use of 6% hydrogen peroxide gel strips.
3.3.10. Photo-induced toxicity
3.3.10.1.Phototoxicity / photoirritation and photosensitisation
No data submitted
3.3.10.2.Phototoxicity / photomutagenicity / photoclastogenicity
No data submitted
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