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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
European Union Risk Assessment Report
Author:
European Chemicals Bureau
Year:
2003
Bibliographic source:
European Union Risk Assessment Report. Hydrogen peroxide; CAS No: 7782-84-1; EINECS No: 231-765-0. Final report, 2003

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Test material

Constituent 1
Chemical structure
Reference substance name:
Hydrogen peroxide
EC Number:
231-765-0
EC Name:
Hydrogen peroxide
Cas Number:
7722-84-1
Molecular formula:
H2O2
IUPAC Name:
Hydrogen peroxide
Test material form:
liquid
Remarks:
colourless
Specific details on test material used for the study:
Purity: 35% (w/w)

Test animals

Species:
mouse
Strain:
other: Swiss OF1/ICO:OF1 (IOPS Caw)
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test animals:
- Source: Iffa Crédo, L'Arbresle, France
- Age at study initiation: approximately 6 weeks
- Weight at study initiation:
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: five per sex in polycarbonate cages
- Diet (e.g. ad libitum): AO4 C pelleted diet (U.A.R., Villemoisson-sur-Orge, France) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

Environmental conditions:
- Temperature (°C): 21 +/- 2
- Humidity (%): 50 +/- 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Water
Details on exposure:
The test substance was administered once by intraperitoneal route using a dose volume of 25 mL/kg, which allowed to test higher doses with less concentrated solutions. The quantitiy of the test substance administered to each animal was adjusted according to the body weight recorded at the time of dosing. The vehicle control animals received the vehicle alone, under the same conditions. The positive control animals received cyclophosphamide, by oral route, at a volume of 10 mL/kg.
Duration of treatment / exposure:
Once by intraperitoneal injection
Frequency of treatment:
Once
Post exposure period:
24 and 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
first cytogenetic test
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
first cytogenetic test
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
first cytogenetic test
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
first cytogenetic test
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
second cytogenetic test
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
second cytogenetic test
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
second cytogenetic test
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
second cytogenetic test
No. of animals per sex per dose:
5
Control animals:
yes
Positive control(s):
Cyclophosphamide, administered by oral route in 10 mL/kg at a dose of 50 mg/kg body weight.

Examinations

Tissues and cell types examined:
For each animal, the micronuclei were counted in 20000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
Details of tissue and slide preparation:
At the time of sacrifce, all the animals were killed after CO2 inhalation in excess. The femurs of the mice were removed and the bone marrow eluted out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were suspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with May-Grünwald-Giemsa. All the slides were coded for scoring.
Evaluation criteria:
A positive response was assumed if a statistically significant increase in the number of micronucleated polychromatic erythrocytes (MPE) when compared to the vehicle group occurred, which doubled the number of MPE of the historical control data, i.e. a number greater than 3.6/1000 PE. The results were considered as negative if the above criteria was not fully met.
Statistics:
The mean number of MPE and the PE/NE ratio from the treated groups were compared to simultaneous vehicle groups. The inter-group comparison was performed using: for MPE the X-square test, for PE/NE ratio the Student's t-test in which p = 0.05 was used as the lowest level of significance.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The mean values of MPE of all groups treated with the test substance were similar to those of their respective controls. A slight, statistically significant increase in the MPE number of the low-dose group after observed after 24 hours was considered as biologically insignificant, because the MPE value was within the range of historical controls, no dose-effect relationship was noted, and the increase was essentially attributed to one animal which had 13 MPE/2000 PE. The PE/NE ratio was statistically significant lower at the three doses at 24 hours and at 250 and 1000 mg/kg after 48 hours, showing that the test substance effectively affected the bone marrow cells.
Due to the marked mortality in the 2000 mg/kg dose group, a second cytogenetic study was carried out. In this second test, no mortality and no clinical signs were observed in the 250 and 500 mg/kg dose groups of both sexes and in the female 1000 mg/kg dose group. After treatment with 1000 mg/kg, one of sixteen males died, which was replaced by one of the supplementary group. Hypoactivity and piloerection was noted in the other treated males at 1000 mg/kg. No macroscopic abnormalities (abdominal cavity) were seen at necropsy at doses of 250, 500 and 1000 mg/kg except for a discolourated spleen in one male at 1000 mg/kg.
The mean values of micronucleated polychromatic erythrocytes were within the historical range in the two vehicle groups.
Cyclophosphamide induced a highly significant increase (p < 0.001) in the number of MPE. In addition, the PE/NE ratio decreased significantly (p < 0.05) showing the toxic effects of the positive control substance to bone marrow cells.

Applicant's summary and conclusion

Conclusions:
Under the study conditions, the substance was not genotoxic in bone marrow erythrocyte micronucleus assay in mice.
Executive summary:

A study was conducted to determine the in vivo genetic toxicity of the substance, according to OECD Guideline 474, in compliance with GLP. The test substance was tested for its potential to induce cytogenetic damage to the bone marrow cells of Swiss OF1 mice. Following preliminary toxicity testing, animals received one intraperitoneal injection of the test substance at concentrations of 0 to 2000 mg/kg bw (first cytogenicity test) or 0 to 1000 mg/kg bw (second cytogenicity test). The positive control animals received cyclophosphamide, by oral route, at a volume of 10 mL/kg. For each animal, bone marrow smears were prepared and the micronuclei were counted in 2000 polychromatic erythrocytes. The polychromatic (PE) to normochromatic (NE) erythrocyte ratio was established by scoring 1000 erythrocytes (PE + NE). The mean number of micronucleated polychromatic erythrocytes (MPE) and the PE/NE ratio from the treated groups were compared to simultaneous vehicle groups. The mean values of MPE of all groups treated with the test substance were similar to those of their respective controls. The mean values of micronucleated polychromatic erythrocytes were within the historical range in the two vehicle groups. Cyclophosphamide induced a highly significant increase in the number of MPE and a decrease in the PE/NE ratio showing the toxic effects to bone marrow cells. Under the study conditions, the test substance was not genotoxic in bone marrow erythrocyte micronucleus assay in mice (European Chemicals Bureau, 2003).

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