Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was carried out under GLP conditions and in accordance with the OECD Guideline for Testing of Chemicals No. 474, 1994.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Hydrogen peroxide, 35% w/w

Test animals

Species:
mouse
Strain:
other: Swiss OF1/ICO:OF1 (IOPS Caw)
Sex:
male/female
Details on test animals 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 was used as the vehicle.
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 routed, 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
Remarks:
Doses / Concentrations:
0, 500, 1000, 2000 mg/kg body weight
Basis:
nominal in water
first cytogenetic test
Remarks:
Doses / Concentrations:
0, 250, 500, 1000 mg/kg body weight
Basis:
nominal in water
second cytogenetic test
No. of animals per sex per dose:
five males, five females
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
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The results of the second cytogenetic study are given in Table 1. The mean values of MPE of all groups treated with hydrogen peroxide 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 hydrogen peroxide effectively affected the bone marrow cells. The clinical signs and mortality noted in the first cytogenicity test are given in Table 2. 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 ne 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 (Table 3).
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.

Any other information on results incl. tables

Table 1: Results of the second cytogenicity study.

Group a)

Dose (mg/kg)

MPE/1000 PE b)

PE/NE ration c)

Time of sacrifice d) (hours)

Mean

SD

Mean

SD

Vehicle

-

0.9

0.9

0.6

0.1

24

Test substance

250

2.2**

1.6

0.5*

0.1

500

1.2

1.0

0.5*

0.1

1000

1.6

1.2

0.5*

0.1

Cyclophosphamide

50

37.6***

8.2

0.5*

0.1

Vehicle

-

1.7

0.8

0.7

0.2

48

Test substance

250

1.9

0.8

0.5*

0.2

500

1.1

0.5

0.6

0.1

1000

1.2

0.8

0.5*

0.1

a) 10 animals (5 males, 5 females) per group; b) based on scoring of 2000 polychromatic erythrocytes; c) based on a total of 1000 erythrocytes (PE + NE); d) time following treatment (intraperitoneal injection, dissolved in water); MPE: micronucleated polychromatic erythrocytes; PE: polychromatic erythrocytes; NE: normochromatic erythrocytes; SD: standard deviation; * p < 0.05, ** p < 0.01, *** p < 0.001 (X-square test for MPE, Student's t-test for PE/NE ratio)

Table 2: Clinical signs and mortality noted in the frist cytogenicity study.

Dose (mg/kg)

Time point

Clinical signs

Males (animals/total number of animals)

Females (animals/total number of animals)

500

2 h – 48 h

None

--

--

1000

2 h

Piloerection

2/10

0/10

24 h – 48 h

None

--

--

2000

2 h

Convulsions before death

5/13

7/13

2 h

Convulsions, hypoactivity

3/8*

--

2 h

Convulsions, piloerection, hypoactivity

3/8*

--

2 h

Piloerection, hypoactivity

2/8*

1/6*

2 h

Hypoactivity

--

5/6*

24 h

None

--

3/6*

24 h

Mortality

1/8*

--

24 h

Piloerection, hypoactivity

5/7**

--

24 h

Hypoactivity

2/7**

3/6*

* Animals remaining after first intraperitoneal injection; ** Animals remaining after second intraperitoneal injection

Table 3: Clinical signs and mortality noted in second cytogenic study.

Solution at 4 % hydrogen peroxide

Dose (mg/kg)

Time point

Clinical signs

Males (animals/total number of animals)

Females (animals/total number of animals)

1000

2 h

Hypoactivity

9/16

--

2 h

Piloerection

3/16

--

2 h

Hypoactivity, piloerection

3/16

--

2 h

Coma

1/16

--

24 h

Mortality

1/16

--

24 h

Piloerection

15/16

--

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Hydrogen peroxide did not reveal any genotoxic potential under the experimental conditions of this test.
Executive summary:

The potential of hydrogen peroxide to induce cytogenetic damage to the bone marrow cells of Swiss OF1 mice was tested in a micronucleus assay in accordance with OECD Guideline No. 474 and under GLP conditions. Preliminary toxicity tests were performed to define the doses to be used in the cytogenetic test. Six groups of 5 male and 5 female mice received the test substance by a single intraperitoneal injection at doses of 250, 500 and 1000 mg/kg. Two groups of 5 males and 5 females received the vehicle (water) alone. One group of 5 males and 5 females was treated with the positive control substance cyclophosphamide administered by a single oral dose of 50 mg/kg. For each animal, bone marrow cell 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). In the two vehicle control groups, the mean values of micronucleated polychromatic erythrocytes (MPE) were in the range of historical controls. Cyclophosphamide induced a highly significant increase in the number of MPE and significantly decreased the PE/NE ratio, indicating the cytotoxicity of the control substance. In all groups treated with hydrogen peroxide, the mean values of MPE were similar to those of their respective vehicle controls. A slight statistically significant increase in the MPE number in the low-dose group after 24 hours was considered as biologically insignificant. A statistically significant decrease in the PE/NE ratio in most treated groups after 24 and 48 hours showed that hydrogen peroxide effectively affected the bone marrow cells. It was concluded from the findings that under the experimental conditions hydrogen peroxide did not induce cytogenetic damage in bone marrow cells of mice when administered by the intraperitoneal route.