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Toxicological information

Toxicity to reproduction

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screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
Justification for type of information:
No valid reproductive toxicity study was located for the target substance, hydrogen peroxide- urea (1:1). However, as hydrogen peroxide- urea (1:1) breaks down to one molecule of each hydrogen peroxide and urea, data from these can be used as source data in a read-across approach.
Regarding hydrogen peroxide it is noted that existing data from old, non-guideline do not indicate undue effects on reproduction and development parameters in mice, rats and rabbits. The summaries and conclusions contained in the EU Risk Assessment Report (ECB, 2003) are presented further below. It is stated in the conclusions that it was consented at the Technical Meeting level to derogate reproduction toxicity screening. “The decision was reached on the presumption that conventional study protocols (e.g. administration in drinking water) were unlikely to show specific embryonal or foetal effects firstly, because it is doubtful whether hydrogen peroxide (as opposed to its degradation products oxygen and water) would reach the foetus and secondly, because local effects in the mother, possibly causing nutritional disturbances and general toxicity, are expected.” (ECB, 2003). Therefore, new animal studies for hydrogen peroxide are not required and technically not feasible.

Copy from EU Risk Assessment Report on Hydrogen Peroxide, section Toxicity for reproduction (ECB, 2003):
There were no reproductive toxicity studies available employing appropriate study methods. 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.

The same researchers (Wales et al., 1959) also demonstrated in an in vitro experiment that rabbit semen was more resistant to exogenous hydrogen peroxide (even 3,000 ppm failed to immobilise the spermatozoa completely) than semen from bull, fowl, dog, ram, mouse and human. Rabbit seminal plasma had a particularly high capacity to decompose hydrogen peroxide, presumably due to catalase.

In another old study, 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.

There is a brief account of experimental studies with hydrogen peroxide involving even observations on reproductive effects (Antonova et al., 1974). Male and female rats were administered hydrogen peroxide daily by gavage at doses of 1/10-1/5 LD50 (which is not specified) for 45 days. At the high dose, females showed modifications of the oestrus cycle and males reduced mobility of spermatozoa, without an effect on the testis weight. In another experiment male and female rats received daily doses of 0.005, 0.05, 0.5, 5, or 50 mg hydrogen peroxide/kg bw by gavage for 6 months, and were mated. Variations of the oestrus cycle in females were observed during treatment at 50 and 0.5 mg/kg but not at 5 mg/kg. Reduced mobility of spermatozoa in males was observed at 50 mg/kg. No changes were found in the morphology and weight of the testes. Among the high dose females, 3/9 produced litters, compared to 7/9 in the control group. In addition, litter size and bodyweight gain of the offspring of the high dose females were reduced relative to those of control females. Due to inadequate reporting the study findings cannot be assessed.
Developmental toxicity
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 was no major effect 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.

Hydrogen peroxide has also been tested, together with 7 organic peroxides, with the three-day chicken embryo air chamber method (Korhonen et al., 1984). The total effective (ED50) dose (including all deaths and malformations) was 2.7 mmol H2O2/egg. In the series of eight peroxides studied, hydrogen peroxide exhibited a low potency of embryotoxicity. Overall, the peroxides were judged not to be (comparatively) very effective in causing malformations.

Conclusions on reproductive toxicity
No appropriate animal studies were available for a complete evaluation of reproductive and developmental toxicity. Two limited studies with mice and rats exposed to hydrogen peroxide in drinking water suggested no grave disturbances on the male or female reproductive functions (Wales et al., 1959; Hankin, 1958). Moreover, an appropriate 90-day drinking water study with catalase-deficient mice (FMC, 1997), and carcinogenicity studies with catalase-deficient mice (Ito et al., 1981a;b) and F344 rats (Takayama, 1980) did not identify testes or ovaries as target organs for toxicity. 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 (the authors proposed that the deleterious effect was due to the breakdown of essential nutrients in food by hydrogen peroxide). Although raising some further questions, the study cannot be used for an evaluation.

Thus there is a clear data gap regarding studies of developmental toxicity for hydrogen peroxide. Industry had however requested a derogation for reproductive toxicity screening which was consented at the Technical Meeting level. The decision was reached on the presumption that conventional study protocols (e.g. administration in drinking water) were unlikely to show specific embryonal or foetal effects firstly, because it is doubtful whether hydrogen peroxide (as opposed to its degradation products oxygen and water) would reach the foetus and secondly, because local effects in the mother, possibly causing nutritional disturbances and general toxicity, are expected.

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Applicant's summary and conclusion