Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997-10-29 to 1997-12-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

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)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
not specified
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mammalian somatic cell cytogenicity assay

Test material

Reference
Name:
Unnamed
Test material form:
solid

Test animals

Species:
mouse
Strain:
other: Albino Crl:CD-1TM (ICR) BR
Details on species / strain selection:
The mouse has been shown to be a suitable model for this type of study was recommended in the test method.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (IUK) Limited, Margate, Kent
- Age at study initiation: five to eight weeks
- Weight at study initiation: 25 to 30 g (male); 20 to 26 g (female)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: in groups up to five in solid-floor polypropylene cages with woodflakes bedding
- Diet (e.g. ad libitum): free access to food (Rat and Mouse Expanded Diet No. 1, Special Diets Services Limited, Wtiham, Essex, UK)
- Water (e.g. ad libitum): free access to drinking water
- Acclimation period: five days at minimum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22°C
- Humidity (%): 59-64 %
- Air changes (per hr): approximately 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours darkness

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle/solvent used: arachis oil
- Justification for choice of solvent/vehicle: The test substance is hydrolytically unstable.
- Concentration of test material in vehicle: 12.5, 25, and 50 mg/mL (main study)
- Amount of vehicle: 10 mL/kg
- Lot/batch no.: T30
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was freshly prepared as required as a suspension at the appropriate concentration in arachis oil.

Duration of treatment / exposure:
single exposure
Frequency of treatment:
Once
Post exposure period:
24 hours after dosing (first group); 48 hours after dosing (second group)
Doses / concentrationsopen allclose all
Dose / conc.:
125 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
No. of animals per sex per dose:
5 animals per sex and dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide;
- Justification for choice of positive control(s): guideline suggestion
- Route of administration: oral
- 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 study was conducted to determine a suitable dose level. The level selected should ideally be the maximum tolerated dose level or that which produces some evidence of cytotoxicity up to a maximum recommended dose of 2000 mg/kg bw.

TREATMENT AND SAMPLING TIMES: Animals were dosed once only via the route with the test material at 125, 250 or 500 mg/kg bw. Sampling was done 24 or 48 hours after treatment.

DETAILS OF SLIDE PREPARATION: Femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grünwald/Giemsa.

METHOD OF ANALYSIS: Stained bone marrow smears were coded and examined 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.


Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythorcytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.

A positive mutagenic response was demonstrated when a statistically significant increase in the number of imcronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.

If these criteria were not demonstrated, then the test material was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.
Statistics:
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a √ (X + 1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250 – 2000 mg/kg bw (ip), 2000 mg/kg bw (oral)
- Clinical signs of toxicity in test animals: none (oral route); premature deaths were observed at and above 1000 mg/kg and clinical signs were observed at and above 250 mg/kg, as follows: hunched posture, lethargy, ptosis, dehydration, ataxia, splayed gait and decreased respiratory rate (ip administration)

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: No statistically significant increases in the frequency of micronucleated PCEs compared to the vehicle control were observed.
- Ratio of PCE/NCE: A statistically significant decreases in the PCE/NCE ratio in the 24-hour 125 and 500 mg/kg bw test material groups compared to the vehicle control were observed.
- Appropriateness of dose levels and route: The maximum tolerated dose (MTD) of the test material selected for use in the main study was 500 mg/kg bw, with 125 and 250 mg/kg bw as the lower dose levels. The intraperitoneal route of administration was selected for use based on the results of the range-finding study.


Applicant's summary and conclusion

Conclusions:
The test material did not produce a significant in crease the frequency of micornuclei in polychromatic erythrocytes of mice under the conditions of the test. Thus, the test material was considered to be non-genotoxic.
Executive summary:

A study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method used has been designed to comply with the UKEMS sub-committee on guidelines for mutagenicity testing, Report, Part 1 revised (Basic Mutagenicity Tests: UKEMS recommended procedures, 1990). The study design also complies with the revised OECD Guidelines for Testing of Chemicals No. 474 "Micronucleus Test", Method B12 of EEC Commission Directive 92/69/EEC, the US, EPA, TSCA and FIFRA guidelines and the Japanese Ministry of International Trade and Industry guidelines for the testing of new chemical substances. Sampling of bone marrow did not occur after 48 hours, the need for the later sampling time (72 hours) is now considered to be unnecessary.

A range-finding study was performed to find suitable dose levels for the test material and route of administration. The micronucleus study was conducted via the intraperitoneal route in groups of ten mice (five males and five females) at the maximum tolerated dose (MTD) 500 mg/kg with 125 and 250 mg/kg as the two lower dose levels. Animals were killed 24 and 48 hours later, the bone marrow extracted and smear preparations made and stained. Polychromatic and normochromatic erythrocytes were scored for the presence of micronuclei.

Further groups of mice were given a single intraperitoneal dose of arachis oil or dosed orally with cyclophosphamide, to serve as vehicle and positive controls respectively. 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. A statistically significant decrease in the PCE/NCE ratio was observed in the 24-hour test material dose groups when compared to their concurrent control groups. This observation and the presence of clinical signs at and above the 125 mg/kg dose level and the premature deaths in the 24 and 48 -hour 500 mg/kg test material dose groups was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

The positive control material produced a marked increase in the frequency of micronucleated polychromatic erythrocytes. The test material, TODI, was considered to be non-genotoxic under the conditions of the test.