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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Korea National Institute of Environmental Research Public Notice No 2006-4, Annex 5 Genotoxicity Test and SOPs of KTR.
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: Solid

Test animals

Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Species: Mouse, ICR (Crl:CD l BR), male
- Source: Orient Incorporation, 699-13 Mokdong-Ri, Buk-Myeon, Gapyoung-Gun, Kyonggi-do, Korea
- Age at study initiation: 6 weeks at reception
- Weight at study initiation: 21.83 to 26.81g
- Assigned to test groups randomly: Yes, stratified random sampling method with the index of animal bodyweight and excluding animals with clinical signs
- Housing: 6 animals / polycarbonate cage
- Diet (e.g. ad libitum): Pelleted food for experimnetal use, (Purina Korea Incorporation, 85-1 Jangdang-Dong, Pyongtaek-Si, Kyonggi-do, Korea) ad libitum
- Water (e.g. ad libitum): Tap water purified by fitration ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 to 70
- Air changes (per hr): 12 to 15 times / hour
- Photoperiod (hrs dark / hrs light): twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Water
Details on exposure:
Groups, each of six male mice, were dosed once only via the intraperitoneal route with the test item at 0, 137.5, 275 or 550 mg/kg. A further group of six males was treated with the positive control Cyclophosphamide.The animals were killed by cervical dislocation 24 hours following treatment.
Duration of treatment / exposure:
24 hours
Frequency of treatment:
Once only
Post exposure period:
24 Hours
Doses / concentrations
Remarks:
Doses / Concentrations:
137.5, 275 or 550 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
6 males / group
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control item was supplied by Sigma Chemical Co, as follows:

Supplier's identification: Cyclophosphamide
Supplier's lot number: 87H0207
Purity: 98%
Storage conditions: Refrigeration 4ºC

For the purpose of this study the positive control item was dosed at 10 ml/kg at a dose level of 70 mg/kg.

Examinations

Tissues and cell types examined:
Mammalian erythrocytes.
Details of tissue and slide preparation:
The animlas were sacrificed 24 hours after administration, the femur was removed and both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone marrow was flushed with foetal bovine serum. The cells were collected to the centrifuge tube and centrifuged at 1000rpm for 5 minutes. The supernatant was discarded and the concentrated cells were suspeneded with a little fresh serum. The cell suspension was smeared on a clean slide. The smeared slide was air dried and therafter fixed for 5 minutes with methanol and stained with 40μg/ml acridine orange.
Evaluation criteria:
Stained bone marrow smears were coded and examined blind using fluorescent microscope at magnification over 400X.
Criteria for judgement
Polychromatic erythrocytes (PCE) are determined by appearing orange fluorescent light without nuclei

Nonchromatic erythrocytes (NCE) are determined by appearing only their black shadows without fluorescent light

Criteria for Micronuclei
-Size : From as small as distinguishable to as large as a half the diameter of erythrocytes
-Shape: mainly round and included the form of a donut shape, half moon shape
-Colour: THe same colour with near cell nucleus: green fluorescent in acridine orange

Observation of Specimen : Micronucleated polychromatic erythrocytes (MNPCE) were counted in 2000 polychromatic erythrocytes per animal. Additionally Polychromatic erythrocyte frequency in 100 whole erythrocytes (PCE+NCE) was calculated
Statistics:
Statistical analyses were determined on the base of P value 0.05 between the vehicle control's micronucleated polychromatic erythrocyes and the test item groups. The determination of dose dependence was by trend test according to the statistical results.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The frequencies of MNPCE were
Negative control = 0.13% ± 0.05%
137.5 mg/kg = 0.14 ± 0.05%
275 mg/kg = 0.13% ± 0.04%
550 mg/kg = 0.16 ± 0.05%
Positive control = 6.76 0.65%
There was no statistically significant difference at any dose level of the test item.
The positive control showed a statistically significant increase (p<0.01)

The ratio of PCE's to total erythrocytes as an index of bone marrow toxicity, were
137.5 mg/kg = 50.81
275 mg/kg = 51.28
550 mg/kg = 50.48
Positive control = 49.38
There was no significant difference in the ratio between any test item treated group and negative control


There were no deaths. However in some test item groups there was a statistically related decrease in bodweights compared to the vehicle control.

Any other information on results incl. tables

See attached background information

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test item was considered to be non-mutagenic under the conditions of the test.
Executive summary:

Introduction. 

The study was conducted to determine if the test material would cause genotoxic changes in chromosomes or the miotic apparatus of murine polychromatic erytjhrocytes The study was conducted in accordance with toxicity test guideline of Korea National institute of Environmnetal Research Public notice No 2006 -4, Annex 5 Genotoxicity test and the method was designed to be compatible with the 1997 OECD Guidelines for Testing of Chemicals No.474 "Mammalian Erythrocyte Micronucleus Test".

Methods. 

The test item was administered once intraperitoneally to groups of 6 male rats at a dose of either 0, 137.5, 275 or 550. Animals were killed 24 hours later, the bone marrow extracted, and smear preparations made and stained. The number of Micronucleated Polychromatic Erythrocytes was counted in 2000 Polychromatic Erythrocytes.

Results. 

There was no statistically significant increase in the frequencies of Micronucleated Polychromatic Erythrocytes at any dose level tested. and no significant difference was observed in the ratio of Polychromatic Erythrocytes to total erythrocytes between the test item groups and the vehicle control.

A statistically significant increase was observed in the positive control group hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Conclusion. 

The test item was considered to be non-mutagenic under the conditions of the test.