Registration Dossier

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
Study period:
September 6, 1983 - November 14, 1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
nr. of animals used and treatment schedule
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Type of assay:
chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Santicizer 141 Plasticizer
- Physical state: liquid
- Analytical purity: confidential
- Lot/batch No.: confidential
- Storage condition of test material: at room temperature
- Stability under test conditions: Under the conditions of the assay the compound was assumed to be stable

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River Breeding Laboratories, Inc., Kingston, New York, USA
- Age at study initiation: Approx. 50 days old
- Weight at study initiation: Males: 303.1 ± 16.3 to 315.6 ± 13.8 grams; Females: 220.1 ± 10.8 to 212.9 ± 9.4 grams
- Assigned to test groups randomly: yes, under following basis: computer-generated random numbers
- Fasting period before study: no data
- Housing: individually in wire-mesh cages
- Diet: Purina Lab Meal #5001 ad libitum
- Water: ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.11 - 23.33
- Humidity (%): 69-83%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Lot/batch no. (if required): 80235
- Purity: 100 %
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was freshly prepared on the day of administration. The dosing solutions were prepared on a weight per volume basis.
Duration of treatment / exposure:
6, 12, 24, and 48 hours
Frequency of treatment:
a single dose at each dose level
Post exposure period:
Post-Treatment Sampling Times (Bone Marrow Cell Harvest): six males and six females from each dose level and the negative control group were sacrificed at 6, 12, 24, and 48 hours in order to sample cells exposed at various points in the cell cycle. The positive control rats were sacrificed 24 hours after treatment.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1500, 5000, and 15000 mg/kg
Basis:
no data
No. of animals per sex per dose:
Test substance and vehicle control: 24; Positive control: 6
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow cells were collected from both femurs of each rat. These cells were processed, stained, and examined micrcscopically. Wherever possible, 60 metaphase cells were examined from five of the six rats per sex per group.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose levels were chosen based on the results of the preliminary range-finding study and the previously determined oral LD.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 6, 12, 24, and 48 hours

DETAILS OF SLIDE PREPARATION: The bone marrow cells were processed according to the modified techniques described by Evans (Evans, H.J. Cytological Methods for detecting Chemical Mutagens; Chemical Mutagenesis; Principles and Methods for Their Detection, Volume 4, ed. by A. Hollaender, Plenum Press, N.Y., pp. 1-30, 1976) and Killian, et al. (Killian, D.J., F.M. Moreland, M.C. Benge, M.S. Legator and E. B. Whorton: A Collaborative Study to Measure Interlaboratory Variation with the In vivo Bone Marrow Metaphase Procedure, Handbook of Mutagen Testing, Elsevier/North-Holland, Amsterdam, pp. 243-260, 1977).
Immediately following sacrifice, bone marrow cells were collected from both femurs of each animal by aspiration. The aspirate was centrifuged and the supernate was decanted and 0.075M KC1 was added to each tube. After 25 minutes, freshly prepared fixative was added to each tube. The tubes were centrifuged and the supernate was decanted and fixative was added again several times. Finally several drops of the final cell suspension were dispersed onto glass microscope slides and air-dried. Two slides were made for each animal. The cells were stained with a Giemsa solution for 10 minutes. Slides were mounted with glass cover slips in mounting media.

METHOD OF ANALYSIS: Chromosome Evaluation: an attempt was made to examine at least 60 cells in metaphase from 5 of the 6 rats chosen randomly for each sex and group. Slides prepared from the 48-hour sacrifice were not analyzed for chromosomal aberrations. Only those cells in the metaphase stage of mitosis were analyzed for the presence of cytogenetic abnormalities. The following items are recorded for each animal: numbers and types (classification) of chromosomal aberrations, mitotic index, modal number for each metaphase and the vernier location of each metaphase containing damage. The mitotic index was recorded as the number of cells undergoing mitosis per 500 cells counted, and is represented as a percentage.
Evaluation criteria:
No data
Statistics:
The mean mitotic indices, mean modal numbers, percent aberrant cells and the mean number of aberrations per cell for each group were statistically compared using the Kruskal-Wallis nonparametric analysis of variance and nonparametric pairwise group comparisons (KW-ANOVA). Body weight data was analyzed by analysis of covariance (ANCOVA). All tests were one-tailed at the 95% confidence interval (p <.05).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1500, 5000 and 15000 mg/kg bw
- Solubility: in corn oil
- Clinical signs of toxicity in test animals: No animals died while on study. Abnormal clinical observations were seen for all three groups treated, increasing in number and severity along with increased dose. Males and females administered all three levels lost weight between Day 0 and Day 1.
- Evidence of cytotoxicity in tissue analyzed: The test compound produced no apparent effects on the mitotic indices of the animals. Based on these results 15000 mg/kg was chosen as the highest dose to be tested for the in vivo Chromosome Study.
- Harvest times: 24 hours

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No significant increases in the number of chromosomal aberrations occurred in the treated groups when compared to the control group.

Any other information on results incl. tables

Soft feces, urine stains, depression, rough coat, red stains on nose and eyes, and hunching were seen with increasing frequency in a dose-related trend. Body weights were significantly depressed in a dose related fashion at 24 and 48 hours.

5 of the 12 rats in the mid dose group (5000 mg/kg bw) died during the second day after dosing.

A statistically significant difference in mitotic indices was seen for the 12 hour, high dose animals (15000 mg/kg bw); 8 of the 12 animals from this group had no analyzable metaphases recovered. It seems unlikely that a toxic effect to the bone marrow would be seen only at 12 hours after treatment, so the reasons for this finding remain unclear.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this in vivo cytogenetics study, Santicizer 141 Plasticizer did not damage chromosomes in rat bone marrow cells even after exposure at dose levels producing toxicity. Therefore, under the conditions of this study, Santicizer 141 Plasticizer is considered not to be clastogenic at any of the levels tested.
Executive summary:

This study was designed to evaluate the clastogenic potential of Santicizer 141 Plasticizer as measured by increases in numerical and structural chromosomal aberrations in rat bone marrow cells from Charles River Sprague-Dawley rats. The study was performed similar to OECD Guideline 475. A single dose of the test material was administered by oral gavage to 4 groups of 24 male and 24 female rats at levels of 0, 1500, 5000, and 15000 mg/kg bw. 6 males and 6 females from each group were sacrificed at 6, 12, 24, and 48 hours after the single administration of the test material. Since no evidence of mitotic delay was seen after analysis of the mitotic indices, the slides from the 48-hour sacrifice were not analyzed for chromosomal aberrations.

The acute oral administration of 1500, 5000 and 15000 mg/kg bw of Santicizer 141 Plasticizer to male and female rats produced no significant increases in the frequency of chromosomal aberrations when compared to the control group.

Soft feces, urine stains, depression, rough coat, red stains on nose and eyes, and hunching were seen with increasing frequency in a dose-related trend. Body weights were significantly depressed in a dose related fashion at 24 and 48 hours. 5 of the 12 rats in the mid dose group (5000 mg/kg bw) died during the second day after dosing. A statistically significant difference in mitotic indices was seen for the 12 hour, high dose animals (15000 mg/kg bw). 8 of the 12 animals from this group had no analyzable metaphases recovered. It seems unlikely that a toxic effect to the bone marrow would be seen only at 12 hours after treatment, so the reasons for this finding remain unclear.

Cyclophosphamide, the positive control compound, caused a highly significant increase in the percentage of cells with aberrations and the number of aberrations per cell. The average number of chromosomes in the examined metaphases was not significantly different in the cells from treated rats. No significant difference was observed between the negative control and test groups when comparing modal numbers.

Under the conditions of this in vivo cytogenetics study, Santicizer 141 plasticizer did not damage chromosomes in rat bone marrow cells even after exposure at dose levels producing toxicity. Therefore, under the conditions of this study, Santicizer 141 Plasticizer is considered not to be clastogenic at any of the levels tested.