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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
1985

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Remarks:
Inveresk Research International
Type of assay:
chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material: Nicotinic acid

Test animals

Species:
rat
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: Male 248-275 g, females 180-223
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: Animals were kept in polypropylene (41 cm x 25 cm x 20 cm) cages with stainless steel tops and mesh floors. The cages were placed upon polypropylene collection trays containing absorbent paper which was changed weekly. The male and female rats were individually housed in the same animal holding room with the 2 sexes kept on separate cage racks throughout the study.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C, range 18 -23 °C
- Humidity: 47 %, range 42 – 54 %
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES
- From: 2 June 1985
- To: 6 June 1985

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Distilled water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item solutions (100 mL in distilled water were freshly prepared immediately before dosing using the following procedure:
20 g P0076D + distilled water + 14.3 ml 10 M NaOH
The resulting 200 mg/mL dose formulation was brought within an acceptable pH range by buffering with the indicated quantity of 10 M NaOH, giving a final pH value of 5.7 in the preparation. The 64 mg/mL middle dose and 20 mg/mL low dose test solutions were then prepared by further dilution of the obtained 200 mg/mL high dose test concentration.
CP was dissolved in distilled water to give 1 mg/mL solutions. In all cases, the dose volume was 10 mL/kg bw/day and the route of administration orally by gavage fitted to a 5 ml plastic syringe. Colchicine was freshly made up in Hank1s balance salt solution (HBSS) and given to the animals by intraperitoneal injection (i.p.) in a standard dose volume of 10 mL/kg.
Duration of treatment / exposure:
In the cytogenetic test rats were dosed for 5 consecutive days, then bone marrow samples were taken. The dose groups were as follows:
Test Group: Treatment (5 day), Sex
Vehicle Control: 10 ml Dist. water/kg bw/day, male and female
Positive Control: 10 mg CP/kg bw/day, male and female
Low Dose: 200 mg/kg bw/day, male and female
Middle Dose: 640 mg/kg bw/day, male and female
High Dose: 2000 mg/kg bw/day, male and female
Animal health status checks were done prior to and immediately after dosing on each of the 5 exposure days. On each day of treatment, the body weights of the test animals were recorded before commencement of dosing.
Frequency of treatment:
Daily
Post exposure period:
Marrow samples were taken 6 hours after last dose.
Doses / concentrations
Remarks:
Doses / Concentrations:
200, 640, 2000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
5 groups of 5 male and 5 female CD rats
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: Orally by gavage
- Doses / concentrations: 10 mg/kg bw/day

Examinations

Tissues and cell types examined:
Rat bone marrow cells
Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Prior to this cytogenetic assay, a toxicity test was conducted by the sponsor and the 5 day oral LD50 value determined as 4000 mg/kg bw. Based on results obtained concentrations of 200, 640 and 2000 mg/kg bw/day were selected for use in the cytogenetic experiment subsequently performed. In the cytogenetic test, 5 consecutive daily doses of the test compound were given orally by gavage to the rats at concentrations of 200, 640 and 2000 mg/kg bw/day corresponding to toxicity fractions of 0.05 x LD50, 0.16 x LD50 and 0.50 x LD50, respectively.

TREATMENT AND SAMPLING TIMES
In the cytogenetic test, 5 groups of 5 male and 5 female CD rats were dosed for 5 consecutive days, then bone marrow samples taken 6 hours after the last dose (day 5).

METAPHASE PREPARATION
Each rat was injected i.p. with 2 mg/kg bw colchicine dissolved in HBSS 2 h before each scheduled kill by neck dislocation, following ether asphyxiation. One femur from each animal was dissected out, cleaned of adherent tissue and the marrow aspirated into a plastic heparinised tube containing HBSS at room temperature.

DETAILS OF SLIDE PREPARATION:
After centrifuging the cells at 1,500 r.p.m. for 5 min and decanting the supernatant fluid, the cells were washed once with HBSS, then treated with 4 ml of 0.075 M KCl at 37°C for 20 min. After further centrifugation, freshly prepared fixative was added (methanol:glacial acetic acid 3:1) to the cell pellet and the suspension homogenised by aspiration with a Pasteur pipette. The fixative was then changed and the cell suspension stored for 3 days at 4°C. Prior to making the slides the fixative was changed once more and 5 slides prepared from each animal. Finally, the air dried slides were stained for 15 min with 10% Giemsa (Gurr R66), dehydrated in a series of alcohol changes and cleared in xylene. Coverslips were sealed with D.P.X. mountant.

METHOD OF ANALYSIS:
Slides were assigned numbers corresponding to the coded tube numbers, unrelated to the animal or group number, and were read 'blind' in a random order. Magnification was nominally xl000 using xl0 magnification eye pieces and a xl00 oil immersion objective. From each animal, 50 metaphases with a minimum of 40 well spread chromosomes were examined and scored. The locations of all abnormal spreads examined were recorded using the microscope stage Vernier scale. The slide number was always located on the right hand side. Scored abnormalities (Gebhart, 1970) were notably gaps, breaks, acentric fragments, exchanges, dicentrics, rings, translocations (within the limitations of the staining methods), pulverisations and multiple aberrations.
Evaluation criteria:
All chromosome aberrations, as well as being recorded separately, were converted into lesions (or break scores) in the following manner:
chromatid and chromosome (isochromatid) gaps, breaks and fragments were designated one lesion each, while markers, i.e. exchanges, rings and dicentrics were counted as 2 lesions each (Bauchinger et al, 1976). Pulverised chromosomes were given the artificial value of 5 lesions, whereas cells with chromosomes showing multiple and extensive damage were awarded the artificial maximum value of 10 lesions per metaphase.
The clastogenic potential of a compound was evaluated by calculating the lesion/cell ratio and the percentages of aberrant cells including and excluding gaps. For comparison, the incidence of metaphases showing heteroploidy, i.e. hypodiploidy (40 or 41 chromosomes), hyperdiploidy (43-45 chromosomes) or polyploidy (>45 chromosomes) was also recorded.
Statistics:
For the statistical analysis, Chi-square and Fischer exact tests with significant levels adjusted for non-independence of the contingency table were carried out (Cox, 1970). The chromosomal parameters examined were:
1) Percent aberrant metaphases including cells with gaps alone
2) Percent aberrant metaphases excluding cells with gaps alone
3) Percent aberrant metaphases with numerical alterations.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Based on toxicity data supplied by the sponsor, it was agreed that compound concentrations of 200, 640 and 2000 mg/kg bw/day would be the exposure levels used in the cytogenetic test. Accordingly, the male and female rats were dosed orally for 5 days with the test substance as follows:
Test Group: Treatment (5 day), Sex
Vehicle Control: 10 ml Dist. water/kg bw/day,
Positive Control: 10 mg CP/kg bw/day
Low Dose: 200 mg/kg bw/day, 0.05 x LD50
Middle Dose: 640 mg/kg bw/day, 0.16 x LD50
High Dose: 2000 mg/kg bw/day, 0.50 x LD50
- Solubility: Soluble up to highest concentrations

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels: No abnormal findings or observations were noted in the course of the 5 day dosing regime. Similarly, no appreciable weight fluctuations were recorded in the various exposure groups, all but one rat (No. 7 female, vehicle control) showing normal gains in body weight.
There was no evidence to suggest increased chromosomal aberration frequencies when bone marrow samples from the male and female rats dosed with P0076D were analyzed for clastogenic damage. The sporadic chromatid and chromosome breaks observed in the test compound dosed rats were not significantly increased over corresponding vehicle control values. Compared to the vehicle control, a marginally elevated frequency of aberrant cells showing breaks and fragments (1.5 %) was noted in the males exposed to 2000 mg/kg bw/day of test substance, but this response was clearly a random event and attributed no clastogenic significance.
In rats treated with the low dose of 200 mg P0076D/kg bw/day, one animal (24, male) showed a Robertsonian translocation and another animal (28, female) contained a single pulverized metaphase. Again, however, these events were not considered indicative of any clastogenic compound activity for the following 2 reasons. First, because both aberration phenomena occur sporadically in rats exposed to vehicle substances alone. And second, because comparative findings were conspicuously lacking from rats given higher concentrations of test item. Hence, these observations from the low dose group were regarded as spurious in origin and of no biological consequence for this assay.
- Statistical evaluation: There was also no evidence (P < 0.05) to show increases in the numerical aberration frequencies when bone marrow from the test item exposed rats was examined for ploidy changes. The marginal increase in hyperdiploidy (HR) noted in the low dose group was not encountered at higher treatment levels of test item and was mainly caused by 2 rats (23 male/3 HR cells; 26 female/2 HR cells) showing slightly elevated numbers of aneuploid metaphases
Animals dosed with the positive control substance, cyclophosphamide, showed large and significant (P < 0.001) yields of chromosome aberrations, both when statistical analysis was performed on the sexes separately (significance for males only), or on the male and female rats combined. The increases consisted exclusively of a greater preponderance of chromatid/ isochromatid simple lesions with no exchange figures or complex aberrations recovered from any of the positive control treated rats.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
There was no evidence to show, in either sex at any dose, that the tes item induced either structural or numerical chromosome aberrations to bone marrow cells when administered orally to CD rats for 5 days using considerable LD50 fractions of the test agent.
Executive summary:

A study was carried out according to EU Method B.11 and OECD Guideline 375 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test). The test item was assessed for cytogenetic effects in young adult male and female CD rats. Prior to this cytogenetic assay, a toxicity test was conducted and the 5 day oral LD50 value determined as 4000 mg/kg bw/day. Compound concentrations of 200, 640 and 2000 mg/kg bw/day were selected for use in the cytogenetic experiment.

In the cytogenetic test, 5 consecutive daily doses of the test compound were given orally by gavage to the rats at concentrations of 200, 640 and 2000 mg/kg bw/day corresponding to toxicity fractions of 0.05 x LD50, 0.16 x LD50 and 0.50 x LD50, respectively. One control group of animals was treated with the vehicle, 10 ml distilled water/kg bw/day alone and another received the positive control substance, cyclophosphamide, at a dose level of 10 mg/kg bw/day.

No significant differences in the chromosomal aberration frequencies between the vehicle control and test compound treated animals were observed. Rats dosed with the positive control substance, cyclophosphamide, showed approximately 4-fold increases in the chromosomal aberration yields.

It was concluded that the test item was not capable of inducing structural or numerical aberrations when tested for such potential in bone marrow cells of male and female CD rats using a 5 day dosing regime.