Reaction mass of copper oxide and manganese dioxide

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

year of publication: 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

not fully conducted according to current guideline (older version), only one sampling time, documentation lacking

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

21 JUL 1997

Deviations:

no

Principles of method if other than guideline:

- Principle of test: Micronucleus Test
- Short description of test conditions: MNT performed according to method of Schmid Mut. Res. (1975), using bone marrow cells extracted from the thigh bone (femur) of rats
- Parameters analysed / observed: scored for the presence of micronuclei

GLP compliance:

not specified

Type of assay:

other: micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Manganese dioxide

EC Number:

215-202-6

EC Name:

Manganese dioxide

Cas Number:

1313-13-9

Molecular formula:

MnO2

IUPAC Name:

Manganese dioxide

Test material form:

solid

Specific details on test material used for the study:

The study investigated the test item in form of nano- and micro-sized particles. The mean size distribution of test item particles were 42.63 ± 23 nm for nano-particles and 2.95 ± 31 µm for micro-particles. Both particles acted comparable in the MNT assay, therefore this record focuses on micro-sized particles only.

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Sigma Chemical Co., St. Louis, USA
- Expiration date of the lot/batch: not specified
- Purity: ≥99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: suspended in Milli Q water


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: suspended various doses of test item in Milli Q water after sonication using a probe sonicator (UPH 100, Germany) for 10 min at 90% amplitude


OTHER SPECIFICS:
- specific surface area analysis was determined by using the Brunauer–Emmett–Teller technique = 7.24 (m2 /g)

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Albino

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Institute of Nutrition, Hyderabad, India
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at delivery: 6 to 8 weeks
- Weight at delivery: 80–120 g
- Fasting period before study: not specified
- Housing: in groups of five in polypropylene cages with a stainless steel top grill
- Diet: commercial pellet diet
- Water: ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55–65
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12 /12

IN-LIFE DATES: From: To: not specified

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Milli Q water

Details on exposure:

- suspended in Milli Q water and then applied
- No information on vehicle volume for dose groups available.

Duration of treatment / exposure:

exposed daily for 28 days by oral gavage

Frequency of treatment:

every day

Post exposure period:

24 hours after

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

5 animals

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): known mutagen
- Route of administration: intraperitoneal (i.p.)
- Doses / concentrations: 40 mg/kg bw; volume was 0.01 ml/kg bw

Examinations

Tissues and cell types examined:

polychromatic erythrocytes (PCEs) were examined and scored for presence of micronuclei

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Selection of highest dose was based on induction of a toxic effect without severe sufferings and mortality, and lowest dose showed slight adverse effects.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
24 h after the last treatment, animals were sacrified, femurs removed, bone marrow was collected and centrifuged.

DETAILS OF SLIDE PREPARATION:
The centrifuged cells were spreaded on slides and dried in humidified air overnight. Afterwards, fixiation with methanol and staining with Giemsa (prepared in PBS) was conducted. According to OECD 474 (version from 1997) the study was performed. For each animal 3 slides were made.

METHOD OF ANALYSIS:
The slides were examined under a microscope (1000x magnification) and 2000 PCEs per animal were randomly selected and scored for existing micronuclei.

Evaluation criteria:

A significant difference to control results in a positive result.

Statistics:

- results were expressed as means ± standard deviation (S.D.)
- statistically significant changes were analyzed using one-way ANOVA
- Multiple comparisons were performed using Dunnett’s test
- statistical significance for all tests was set at a p-value below 0.05
- for calculations Graph Pad Instat 3 software for Windows was used

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Remarks:

At highest dose rats showed dullness, irritation and moribund symptoms (no mortality), reduction in body weight gain and feed intake (but loss was not significant)

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The animals, which were treated with 1000 mg/kg bw/d, showed a significant increase in the frequency of micronuclei-PCEs compared to vehicle control. Compared to controls, MnO2-treated groups induced a slight decrease in calculated %PCEs, which indicates the occurrence of cell death. The authors of the study concludes that a possible explanation might be the existence of clastogenic effects, which are involved in micronuclei formation.

Applicant's summary and conclusion

Conclusions:

The repeated daily exposure to manganese dioxide for 28 days, showed in a micronucleus test a statistically significant increase in polychromatic erythrocytes at the highest dose level compared to negative control. Therefore manganese dioxide was considered to be genotoxic.

Executive summary:

In a micronucleus test comparable to OECD guideline 474, the manganese dioxide was administered to male and female Wistar rats (5 per dose) orally by gavage at various doses (0, 30, 300, and 1000 mg/kg bw/d) daily for 28 d and bone marrow cells extracted from the femurs were collected 24 after the last treatment. For each animal three slides were prepared and thereof a total of 2000 polychromatic erythrocytes were scored for the presence of micronuclei. Both controls, negative and positive (cyclophosphamide), were valid. A statistically significant increase in polychromatic erythrocytes compared to negative control was seen at the highest dose level, indicating a genotoxic effect of manganese dioxide.