Registration Dossier

Administrative data

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 15/06/1983 to 21/10/1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No GLP, not specified guidelines, but accurate description of methods and treatment steps.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1983
Report Date:
1983

Materials and methods

Test guideline
Qualifier:
no guideline available
GLP compliance:
no
Remarks:
evidence of a Quality System, but no evidence of a GLP one.
Type of assay:
chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): T2025, compound 38502
- Physical state: clear, colorless non viscous liquid
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory, NY
- Age at study initiation: 6-8 weeks old
- Weight at study initiation: male 208-279 grams, female 140-185 grams
- Assigned to test groups randomly: based on a computer-generated random number table.
- Housing: housed up to three per cage during the quarantine period and singly thereafter in plastic autoclavable cages with wire lids.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: quarantined for 10-14 days after receipt

ENVIRONMENTAL CONDITIONS
- Temperature : 74 ± 6°F
- Humidity (%): 50±20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn Oil, U.S.P. grade, lot A11C, from Eastman C hemical Company, Rochester, NY (carrier vehicle for test article)
Details on exposure:
All rats in the experimental and control groups were weighed immediatley prior to dose administration and the dose volume based on individual body weights. Colchicine, used to arrest dividing cells at metaphase, was administered IP at 1 mg/kg to all rats two to four hours prior to sacrifice.
Two to four hours after the injection of colchicine, the animals were sacrificed by carbon dioxide asphyxiation. Immediately following sacrifice, the femur was exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing HBSS. The bone marrow cells were transferred to a capped centrifuge tube containing 5 ml cold HBSS. The cells were mixed well and maintained in an ice bath until the bone marrow cells had been collected from all the animals.
After centrifugation at approx. 100 x g for 10 minutes, the supernatant fluid was discarded, the cells were resuspended in 5 ml of 37°C 0.075 M KCl and incubated at 37°C for 10 minutes. The cells were collected by centrifugation and resuspended in 5 ml freshly prepared Carnoy's fixative (methanol: acetic acid, 3:1 v/v). After 30 minutes, the cell suspensions were centrifuged, the supernatant liquid discarded, and 5 ml fresh fixative was added to each tube. The tubes were capped and stored overnight at approx. 4°C.
Duration of treatment / exposure:
single administration
Frequency of treatment:
single administration
Post exposure period:
6, 24 and 48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5 ml/kg
Basis:
other: full strenght
Remarks:
Doses / Concentrations:
1.7 ml/kg
Basis:
other: mixed with corn oil
Remarks:
Doses / Concentrations:
0.5 ml/kg
Basis:
other: mixed with corn oil
No. of animals per sex per dose:
5 per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Triethylemelamine (TEM), CAS 51-18-3, lot 02031, obtained from Polysciences, Inc., Warrenton, PA, and dissolved in sterile phosphate buffered saline.
Administered by gavage at a dose level of 4.0 mg/kg.

Examinations

Tissues and cell types examined:
Bone marrow.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Dose-finding study: test article administered by gavage to male and female rats at six dose levels: 15, 10, 5, 2.5, 1 and 0 ml/kg b/w. The test article-vehicle mixture and the vehicle alone were administered by gavage at a rate of 5 ml/kg bw. No animal died at 5 ml/kg or less. Based on these findings, 5 ml/kg was selected as the high dose for the acute cytogentics assay. This dose was selected as the highest dose which could be given without mortality reducing mean body weight by more than 20% or resulting in mortality.

DETAILS OF SLIDE PREPARATION:
To prepare slides, the cells were centrifuged at approx. 100 x g for 10 minutes, the supernatant liquid was decanted, and the cells were resuspended to opalescence in about 1 ml fresh fixative. two to three drops of the cell suspension were dropped onto the center of a cold wet glass slide and allowed to air dry. At least three slides were prepared from each animal. The dry slides were stained with 4% Giemsa stain and permanently mounted. Stained slides were coded and were scored without regard to treatment group. Where possible, a minimum of 50 metaphase cells from each animal was examined and scored for chromatid and chromosome gaps and breaks, fragments, structural rearrangements, and ploidy (1-3).

METHOD OF ANALYSIS: The XY coordinates for each cell with chromosomal aberrations was recorded using a calibrated microscope stage. The ratio of the number of cells in mitosis per 500 cells counted x 100 is defined as the mitotic index. For the purpose of analysis, males and females receiving the same dose level were combined.

Evaluation criteria:
The percentage of cells in the negative control group demonstrating aberrations of any type, other than gaps, must not exceed 4%. The percentage of cells with aberrations in the positive control group must be statistically increased ( p≤ 0.05) relative to the vehicle control using Chi-square statistics.
Statistics:
The t-test was used to compare pairwise the number of aberrations per cell of each treatment group with that of the vehicle control. Each comparison was considered to be between two independent, random samples of unequal variance and a significant increase in the treatment mean relative to the vehicle control (one-sided) was sought.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Doses: 0, 1, 2.5, 5, 10 and 15 ml/kg
- At 15 ml/kg, mortality was 100% for females and 40% for males.
- At 10 ml/kg, mortality was 80% for females and 40% for males.
- No animal died at 5 ml/kg or less. Based on these findings, 5 ml/kg was selected as the high dose for the acute cytogenetics assay. This dose was selected as the highest dose which could be given without mortality reducing mean body weight by more than 20% or resulting in mortality.

There was no apparent change in ploidy in the treatment groups.

TEM induced an average of 0.43 aberrations per cell with 11% of all cells analyzed containing one or more aberrations.

There were slight differences between the pretreatment body weights and those at the time of sacrifice ( <15%).

Clinical signs of toxicity, which were recorded on the day following dose administration, included excess lacrimation and salivation in male and female rats receiv ing 5 ml/kg. Male and female animals receiving 5 and 1.7 ml/kg appeared lethargic. Animals receiving 0.5 ml/kg or corn oil alone appeared normal. No adverse effect registered for TEM administration.

Any other information on results incl. tables

Table 1: Preliminary Acute Dose Range Finding Study

TREATMENT

(ml/kg)

SEX

MORTALITY

0

M

0/5

F

0/5

1

M

0/5

F

0/5

2.5

M

0/5

F

0/5

5

M

0/5

F

0/5

10

M

2/5

F

4/5

15

M

2/5

F

5/5

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The negative and positive controls fulfilled the requirements for determination of a valid test. Under the conditions of this study, T2025 was negative in the acute cytogenetics assay using male and female Sprague-Dawley rats.
Executive summary:

Male and female Sprague-Dawley rats were exposed by gavage to 5.0, 1.7 and 0.5 ml/kg of test article. Bone marrow cells, arrested in metaphase and collected 6, 24 and 48 hours after the single treatment, were examined microscopically for numerical and structural chromosome aberrations. the results of the assay indicate that under the conditions described in this study, the test article did not induce chromosome aberrations in male and female rats.