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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
Study period:
22 Aug 2007 - 21 Sep 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP - Guideline study
Justification for type of information:
refer to category justification report provided in IUCLID section 13

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, London, UK
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
mouse
Strain:
other: Crl:CD1TM (ICR)BR (sufficient albino)
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, UK
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: 23 - 30 g
- Assigned to test groups randomly: yes
- Housing: up to 7 animals per cage in solid-floor polypropylene cages with wood-flake bedding
- Diet: Certified Rat and Mouse Diet Code 5LF2, IPS Ltd., London, UK, ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 12 Sep 2007 To: 14 Sep 2007

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Lot/batch no.: P72
- Concentration of test material in vehicle: 2000 mg/kg bw
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The dosing solution was prepared freshly by dissolving appropriate amounts of the test material in arachis oil yielding a suspension with the final concentration of 2000 mg/kg bw.

Duration of treatment / exposure:
24 and 48 h (vehicle control and dose group)
24 h (positive control group)
Frequency of treatment:
single treatment
Post exposure period:
24 and 48 h (vehicle control and dose group)
24 h (positive control group)
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:

No. of animals per sex per dose:
7 (vehicle control and dose group), 5 (positive control)
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
Tissue: bone marrow
Cell type: polychromatic and normochromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Range finding study performed to find the maximum tolerated dose

DETAILS OF SLIDE PREPARATION: Slides were fixed with methanol and stained with May-Grünwald/Giemsa solution.

METHOD OF ANALYSIS: Slides were examined blind using a light microscope at 1000 fold magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes was scored per animal. In addition, the number of normochromatic erythrocytes was evaluated per 1000 erythrocytes which were also scored for the evaluation of the incidence of micronuclei.

The ratio of polychromatic and normochromatic erythrocytes was calculated.

Evaluation criteria:
For the analyses, the number of micronucleated polychromatic erythrocytes was compared between the test group and the vehicle control group.

MUTAGENICITY
A response would be considered as mutagenic when a statistically significant, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48 h exposure times in regard to the respective vehicle control.
If these criteria were not met, the test substance was considered as non-mutagenic under the applied test conditions.

CYTOTOXICITY
Bone marrow toxicity was considered when the polychromatic to normochromatic ratio was statistically significantly lower in the dose group than in the concurrent vehicle control group.
Statistics:
Mean values and standard deviations were calculated. Statistical analyses were performed by Student´s t test (two-tailed) after √(x + 1) transformation. Any significant result was confirmed via One-Way Analysis of Variance.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 - 2000 mg/kg bw
- Clinical signs of toxicity in test animals: No clinical signs of overt toxicity or mortalities were observed in orally or intraperitoneally exposed animals.
- Other: No marked differences in toxicity were observed in male and female animals.

No evidence of toxicity was observed in animals dosed with the test material via the oral route. Therefore, systemic absorption cannot be confirmed using this dose route.

With no evidence of toxicity being observed in male or female animals dosed with the maximum recommended dose level of 2000 mg/kg bw via the oral and intraperitoneal route, it was considered appropriate to use the intraperitoneal route in the main test to maximize test material exposure and to use male animals only for the main test.

Any other information on results incl. tables

Table 1: Results of the in vivo micornucleus assay in male animals

Treatment group

Number of animals

PCE/NCE ratio at sampling time

 

Total micronuclei per 2000 PCEs at sampling time

 

24 h

48 h

24 h

48 h

Vehicle control (arachis oil)

7

0.77 ± 0.2

0.91 ± 0.42

0.7 ± 0.5

1.1 ± 1.2

Test item (2000 mg/kg bw)

7

0.71 ± 0.26

0.93 ± 0.41

0.6 ± 0.8

0.3 ± 0.5

Cyclophosphamide (50 mg/kg bw)

5

1.23 ± 0.26

 

51.8 ± 15.7***

 

 

PCE: polychromatic erythrocytes

NCE: normochromatic erythrocytes

***: p < 0.001

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative