Registration Dossier

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)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle:
- Amount of vehicle (if gavage or dermal):
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required):
- Purity:
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Duration of treatment / exposure:
A single gavage dose was given.
Frequency of treatment:
A single gavage dose was given.
Post exposure period:
Animals sacrificed at either 24 or 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 200, or 400 mg/kg
Basis:
nominal conc.
400 mg/kg MTD based on preliminary test
No. of animals per sex per dose:
5 animals per sex per treated dose group, vehicle control group, positive control group, and reserve group dosed at high dose.
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: same as treated animals - oral gavage
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
Femoral bone marrow prepared as slides; polychromatic erythrocytes, micronucleated PCEs, total erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: MTD was determined to be 400 mg/kg based on mortality and clinical signs seen at 600 mg/kg in a preliminary study, and LD50 estimate of 550 mg/kg in previous toxicity study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Single gavage, with sacrifice at 24 or 48 hours. Femurs exposed, cut above the knee, and bone marrow aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a labelled centrifug tube containing about 1 ml fetal bovine serum. The cells were pelleted by centrifugation at about 100 g for five minutes and supernatent drawn off. the cells were resuspended.

DETAILS OF SLIDE PREPARATION: A drop of bone marrow suspension was spread on a clean glass slide, affixed with a computer generated label with experiment and animal number. Two slides were prepared per animal. Slides were air dried and fixed in methanol. One set of sldes was stained with a nucleic acid-specific stain (acridine orange) and used for microscopic evaluation.

METHOD OF ANALYSIS: Slides were coded by random number table by person not involved in scoring. Using a fluorescent microscope and medium magnification (400x; blue excitation filter in range of 440 to 490 nm and barrier filter combination at 520 nm0, an area of acceptable quality was selected so that the cells were well spread and stained. Using oil immersion (1000x), the following cellular components were evaluated and enummerated: polychromatic erythrocytes (PCEs), normochromatic erythrocytes (NCEs), micronuclei (M), and the PCEs/ECs ratio.

OTHER:

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
tested at MTD
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes

Any other information on results incl. tables

Summary of Bone Marrow Micronucleus Analysis

Treatment

(mg/kg)

Sex

Hours

Group

Size

PCE/Total erythrocytes (Mean +/- SD)

Change from control (%)

MPCE/1000 PCE (Mean +/- SD)

MPCE/ PCE scored

Vehicle

M

F

24

24

5

5

0.591 (0.06)

0.573 (0.05)

- - -

- - -

0.4 (0.42)

0.2 (0.27)

4/10000

2/10000

 SD10

100

M

F

24

24

5

5

0.538 (0.06)

0.539 (0.06)

-9

-6

0.2 (0.27)

0.2 (0.27)

2/10000

2/10000

SD10

200

M

F

24

24

5

5

0.562 (0.08)

0.486 (0.06)

-5

-15

0.2 (0.27)

0.0 (0.0)

2/10000

0/10000

SD10

400

M

F

24

24

5

5

0.533 (0.06)

0.563 (0.05)

-10

-2

 

 

0/10000

2/10000

CP

50

M

F

24

24

5

5

0.443 (0.05)

0.422 (0.05)

-25

-26

9.3 (0.57)

11.4 (0.82)

*93/10000

*114/10000

Vehicle

M

F

48

48

5

5

0.530 (0.02)

0.530 (0.05)

- - -

- - -

 

0.0 (0.0)

0/10000

0/10000

SD10

400

M

F

48

48

5

5

0.486 0.06)

0.584 (0.06)

-8

10

0.1 (0.22)

0.1 (0.22)

1/10000

1/10000

  • Statistically significant increase compared to vehicle control p +/< 0.05 (binomial distribution, Kastenbaum-Bowman tables)
  • PCE: Polychromatic Erythrocytes      MPCEs: micronucleated polychromatic erythrocytes

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Test article was negative in the rat micronucleus assay because it did not induce a significant increase in micronucleated polychromatic erythrocytes in bone marrow of male and female Hsd:SD rats given a single oral gavage dose at maximum tolerated dose and lower.
Executive summary:

The test article was evaluated for genotoxicity potential (clastogenicity/aneugenicity) as measured by its ability to increase the incidence of micronucleated polychromatic erythrocytes (MPCEs) in bone marrow of male and female Hsd:SD rats. Methods complied with OECD Guideline 474 (Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test). Test article was administered orally by single oral dose gavage of a fomulation in arachis (peanut) oil, at a dose volume of 10 mL/kg. Vehicle control was the arachis oil, and positive control substance was cyclophosphamide monohydrate (50 mg/kg).

           The test was conducted in a dose range finding phase (toxicity) and a definitive phase (micronucleus test). The dose range find study consisted of exposing 5 rats per sex per group to 300, 400, or 600 mg/kg test article. Based on mortality and clinical signs, 400 mg/kg was determined to be the maximum tolerated dose. Mortality was one of five males and two of five females dosed at 600 mg/kg. Animals dosed at 600 mg/kg showed piloerection and lethargy, as well as crusty noses, diarrhea, prostration and hunched posture. Pilorection only was noted in the 300 mg/kg group animals, while the animals in the 400 mg/kg group had piloerection and lethargy. No appreciable reduction in mean group body weights was noted in the study period.

           The definitive study consisted of 7 groups with 5 rats per sex in each. Five of the groups were dosed with control (vehicle or positive) substances or with the test article at 100, 200, or 400 mg/kg, and were euthanized at 24 hours post dose. Rats in the two remaining groups were dosed with either vehicle or the high dose test article formulation and euthanized at 48 hours. An additional 5 rats/sex were dosed at the high dose to be used as replacement animals if needed, but mortality did not occur, and bone marrow was not collected from that group. At the scheduled time point, femoral bone marrow was collected, and marrow smears (slides) were prepared and stained with a nucleic acid specific stain (acridine orange stain). Bone marrow cells (polychromatic erythrocytes (2000 PCEs/animal)] were examined for presence of micronuclei (micronucleated PCEs, MPCEs). Statistical analysis of the data was performed using the Kastenbaum-Bowman Tables (binomial distribution, p </= 0.05).

           No mortality was noted in the definitive study. Piloerection was noted in the 400 mg/kg group animals, and lethargy was noted in the 400 mg/kg group sacrificed at 48 hours. Reductions of up to 15% in the PCEs/ECs ratio of polychromatic erythrocytes to total erythrocytes relative to the vehicle control groups was observed in some of the test groups without a dose response, suggesting that the substance did not markedly inhibit erythropoiesis. No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test substance groups relative to the respective vehicle control groups was observed in male or female rats at 24 or 48 hours post dose administration. (p > 0.05, binomial distribution, Kastenbaum-Bowman Tables). The positive control substance (cyclophosphamide monohydrate) induced a statistically significant increase in the incidence of micronucleated PCEs in male and female rats (p < 0.05, binomial distribution, Kastenbaum-Bowman Tables). The number of MPCEs in the vehicle control groups did not exceed historical control vehicle ranges. All criteria were met for a valid test.

           Under the conditions of the study, a single oral dose of test article at doses up to and including a dose of 400 mg/kg (MTD) did not induce a significant increase in micronucleated polychromatic erythrocytes in bone marrow of male and female Hsd:SD rats. Therefore, the test article was concluded to be negative in the rat micronucleus assay.