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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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
other: UKEMS Sub-committee on Guidelines for Mutagenicity Testing, Report, Part 1 revised (Basic Mutagenicity Tests: UKEMS recommended procedures, 1990).
Qualifier:
according to
Guideline:
other: USA EPA, TSCA and FIFRA guidelines and the Japanese METI/MHLW guidelines for testing of new chemical substances.
GLP compliance:
yes (incl. certificate)
Remarks:
Safepharm Laboratories Limited, Shardlow Business Park London Road, Shardlow Derbyshire, DE72 2GD
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- State of aggregation: liquid
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, in the dark

Test animals

Species:
mouse
Strain:
other: Crl:CD-l™(ICR)BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: approximately five to eight weeks old
- Weight at study initiation: 25 to 30 g
- Assigned to test groups randomly
- Fasting period before study: no
- Housing: up to seven in solid-floor polypropylene cages with woodflakes bedding
- Diet: ad libitum (Certified Rat and Mouse Diet 5LF2, International Product Supplies Limited, Wellingborough, Northants, UK)
- Water: ad libitum
- Acclimation period: minimum of 7 days

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

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle/solvent used: arachis oil
- Concentration of test material in vehicle: 200, 100, 50 mg/mL
Duration of treatment / exposure:
24 or 48 hours
Frequency of treatment:
once
Post exposure period:
not applicable
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
7
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral: gavage
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A range-finding toxicity study was performed to determine a suitable dose level for the micronucleus study.The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg bw.

DETAILS OF SLIDE PREPARATION: The smears were air-dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.

METHOD OF ANALYSIS: Stained bone marrow smears were coded and examined blind using light microscopy at x 1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes were counted; these cells were also scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

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: 2000 mg/kg
- Clinical signs of toxicity in test animals: none
- Evidence of cytotoxicity in tissue analyzed: no

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.
- Ratio of PCE/NCE: There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test material groups when compared to their concurrent vehicle control groups.

Any other information on results incl. tables

EXPERIMENTAL RESULTS

Number of PCE with micronuclei per 2000 PCE PCE/NCE ratio
Treatment Group Group mean SD Group mean SD
negative control, 48 hour sampling time 1.7 1.5 0.81 0.17
negative control, 48 hour sampling time 1.4 0.5 0.82 0.21
positive control, 24 hour sampling time 43.8*** 27.3 1.3 0.28
test article, 2000 mg/kg, 48 hour sampling time 1.1 1.6 0.98 0.27
test article, 2000 mg/kg, 24 hour sampling time 0.4 0.5 0.88 0.36
test article, 1000 mg/kg, 24 hour sampling time 2.6 1.7 0.77 0.32
test article, 5000 mg/kg, 24 hour sampling time 1.4 1.3 1.13 0.37

PCE = Polychromatic erythrocytes

NCE = Normochromatic erythrocytes

SD = Standard deviation

*** = P < 0.001

Applicant's summary and conclusion

Conclusions:
The test material was considered to be non-genotoxic under the conditions of the test.
Executive summary:

The test article’s potential to produce damage to chromosomes or aneuploidy when administered to mice was assessed in a micronucleus assay following OECD guideline 474 and in compliance with GLP. A range-finding study was performed to find suitable dose levels of the test material and investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in test material toxicity between the sexes, therefore the main study was performed using only male mice. The micronucleus study was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum recommended dose (2000 mg/kg bw) with 1000 and 500 mg/kg bw as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single intraperitoneal dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48-hour test material dose groups when compared to their concurrent control groups. There was no evidence of a significant increase in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test material when compared to the concurrent vehicle control groups. The positive control material induced a marked increase in the frequency of micronucleated polychromatic erythrocytes and the test system was therefore considered to be functional. In conclusion, the test material was considered to be non-genotoxic under the conditions of the test.