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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
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EPA OPP 84-2
GLP compliance:
yes
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
-Purity: 96.8%

Test animals

Species:
rat
Strain:
other: Crl:CD(BR)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Kingston, N.Y.
- Age at study initiation: 58 days old
- Weight at study initiation: Males: 237 g (range 217-254 g) and Females: 186 g (range 168-200 g)
- Assigned to test groups randomly: Yes, under following basis:computer-generated random numbers
- Fasting period before study: No data
- Housing: Individually in standard wire mesh cages
- Diet (e.g. ad libitum): Purina Certified Rodent Chow®, Lot Nov 14 851 C)
- Water (e.g. ad libitum): No data regarding the source of the water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.7-22.2°C
- Humidity (%): 40-42%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12-hr light/dark cycles

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn oil
Duration of treatment / exposure:
Single gavage dose
Frequency of treatment:
Single gavage dose
Post exposure period:
animals were sacrificed 6, 24, or 48 hours later for bone marrow sampling.
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 500 mg/kg bw (total dose)
Dose / conc.:
5 000 mg/kg bw (total dose)
No. of animals per sex per dose:
15 males and 15 females
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes, cyclophosphamide - 20 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):6, 24, and 48 hours after administration

DETAILS OF SLIDE PREPARATION: Bone marrow cells were processed according to the procedure of Kilian, et al (1977), with minor modifications. Immediately following sacrifice, the marrow from both femurs of each animal was aspirated into 5 mL pre-warmed (37°C) Hanks' Balanced Salt Solution. The marrow button was collected by centrifugation (IEC Centrifuge Model HNS-II) at approximately 1000 rpm for 5 min and then resuspended in pre-warmed (37°C) 0.075M KCl. After incubation for 15 min in a 37°C waterbath, the centrifugation step was repeated and the resulting pellet resuspended in 5 mL cold fixative (methanol: acetic acid, 3:1). Two or more additional centrifugations and fixative washes were done. After the final centrifugation, the pellet was resuspended in approximately 1.0 mL of fixative. Drops of the cell suspension were dispersed onto pre-cleaned glass microscope slides (wetted in ice-chilled distilled water) and flame-dried. Four slides per animal were prepared. The slides were stained for 7 min with 10% Giemsa (Harleco) in phosphate buffer (pH 6.8), and rinsed with distilled water. After clearing in xylene for 5 min, the slides were coverslipped with Permount®.

METHOD OF ANALYSIS: Representative slides from each animal were scored in a blind manner by H & W Cytogenetic Services, Inc. Chromosomes were examined under high power (1000X) using an oil immersion objective. Fifty metaphase cells per rat were analyzed for chromosomal aberrations. The following observations were recorded for each animal:

• Number and type of structural chromosomal aberrations, and the vernier locations of any abnormal metaphases.

• Number of chromosomes in each cell. The modal number of chromosomes in the rat is 42.
Cells having 40-44 chromosomes were judged acceptable for scoring.

• The mitotic index, as determined by counting the number of mitotic cells among 500 total
cells in the optic fields scanned.
Evaluation criteria:
Chromosomal aberrations were characterized as follows: a) Chromatid-type Aberrations: Damage expressed as breakage of single chromatids or breakage and reunion between chromatids.
These aberrations included: chromatid breaks (tb), isochromatid breaks (ib), fragments (f), triradials (tr), quadriradials (qr)
intrachanges (int), and cells having 10 or more aberrations (≥ 10)

b) Chromosome-type Aberrations: Changes which result from damage expressed in both chromatids at the same site. These aberrations
included: acentric fragments (af), double minutes (dm), ring chromosomes (r), translocations (t), dicentric chromosomes (d), pulverized chromosomes (pu), and pulverized cells (pc)
c) Chromatid (tg) and Isochromatid (ig) Gaps. These were recorded but not scored as aberrations.
Statistics:
For all endpoints, each treated group was compared with the respective negative control group (6, 24, or 48 hours); the positive
indicator group was compared to the 24-hour negative controls. Each animal was considered an experimental unit and treatment effects were examined for each sex independently. For each time point, Mann-Whitney U tests (Conover, 1971) were conducted between the negative control and the treated groups for the analysis of percent abnormal cells, percent abnormal cells with more than one aberration, and aberrations per cell.
In cases where 75% or more of the observations were the same (e.g., where none of the animals in a group had cells with more than one aberration), Fisher exact tests (Conover, 1971) were substituted for Mann-Whitney U tests. Jonckheere tests for trend (Jonckheere, 1954) were also conducted.

Mitotic indices and body weight changes were tested using analysis of variance (Snedecor and Cochran, 1980). Where the F-test for treatment
level was significant, pairwise comparisons were made between each treatment group and the control using Dunnett's test (Steel and Torrie,
1980). Significance was judged at the 5% level.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes

Any other information on results incl. tables

Table 1A Summary of aberration data - 6-hour sacrifice

 

Treatment

Sex

Number of animals per group

Number of Metaphases Analyzed per Group

Percent Abnormal Cells per Group

Percent Cells per Group with > 1 Aberration

Average Number of Aberrations per Cell

Average Number of Mitoses per 500 cells (S.E.)

Corn oil

M

F

5

5

250

250

1.2

0.4

0.0

0.0

0.012

0.004

17.8 (1.3)

18.6 (1.4)

500 mg/kg

M

F

5

5

250

250

0.0

0.0

0.0

0.0

0.000

0.000

12.4 (1.5)

12.2 (2.5)

1500 mg/kg

M

F

5

5

250

250

0.4

0.4

0.0

0.0

0.004

0.004

10.2** (1.8)

12.4 (1.8)

5000 mg/kg

M

F

5

5

250

250

0.0

0.0

0.0

0.0

0.000

0.000

7.6*** (1.3)

11.0* (1.6)

 

* p < 0.05

** p < 0.01

*** p < 0.001

Table 1B Summary of aberration data - 24-hour sacrifice

 

Treatment

Sex

Number of animals per group

Number of Metaphases Analyzed per Group

Percent Abnormal Cells per Group

Percent Cells per Group with > 1 Aberration

Average Number of Aberrations per Cell

Average Number of Mitoses per 500 cells (S.E.)

Corn oil

M

F

5

5

250

250

0.0

0.4

0.0

0.0

0.000

0.004

9.2 (1.0)

7.4 (1.8)

500 mg/kg

M

F

5

5

250

250

0.0

0.0

0.0

0.0

0.000

0.000

6.6 (0.8)

10.6 (1.5)

1500 mg/kg

M

F

5

5

250

250

0.0

0.0

0.0

0.0

0.000

0.000

9.6 (0.6)

14.4 (1.2)

5000 mg/kg

M

F

5

5

250

250

0.0

0.0

0.0

0.0

0.000

0.000

10.2 (1.2)

10.0 (1.1)

 

20 mg/kg Cyclophosphamide

M

F

5

5

250

250

18.4***

33.2***

16.0***

27.2***

1.108***

1.744***

8.8 (1.5)

6.0 (1.8)

 

*** p < 0.001

Table 1C Summary of aberration data - 48-hour sacrifice

 

Treatment

Sex

Number of animals per group

Number of Metaphases Analyzed per Group

Percent Abnormal Cells per Group

Percent Cells per Group with > 1 Aberration

Average Number of Aberrations per Cell

Average Number of Mitoses per 500 cells (S.E.)

Corn oil

M

F

5

5

250

250

0.4

0.0

0.0

0.0

0.004

0.000

9.8 (1.1)

10.6 (1.3)

500 mg/kg

M

F

5

5

250

250

0.0

0.0

0.0

0.0

0.000

0.000

9.4 (1.5)

12.4 (1.5)

1500 mg/kg

M

F

5

5

250

250

0.0

0.0

0.0

0.0

0.000

0.000

7.4 (0.9)

8.6 (2.2)

5000 mg/kg

M

F

5

5

250

250

0.0

0.0

0.0

0.0

0.000

0.000

9.2 (1.9)

8.0 (1.8)

Applicant's summary and conclusion

Conclusions:
Under the conditions of this assay, the test substance is non-clastogenic.
Executive summary:

The test substance was tested for its clastogenic potential in male and female rat bone marrow cells following single acute exposures (500, 1500 or 5000 mg/kg body weight) by oral intubation. Metaphase cells collected 6, 24 and 48 hours after dosing showed no statistically significant increase in structural chromosomal aberrations. Under the conditions of this assay, the test substance is non-clastogenic.