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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study, comparable to guideline study with acceptable restriction

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
yes
Remarks:
dose levels: only one dose evaluted (no limited test because of signs of toxicicty and death observed below 2000 mg/kg bw), only 50 cells/metaphases analysed per animal
GLP compliance:
yes
Type of assay:
chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Santoflex 14

Test animals

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

Administration / exposure

Route of administration:
oral: gavage
Duration of treatment / exposure:
sampling time: 6 h, 18 h and 30 h
Frequency of treatment:
once
Post exposure period:
sampling time: 6 h, 18 h and 30 h
Doses / concentrations
Remarks:
Doses / Concentrations:
1100 mg/kg bw
Basis:

No. of animals per sex per dose:
10 per dose and sampling time (5 males and 5 females)
Control animals:
yes

Examinations

Tissues and cell types examined:
bone marrow cells from both femurs

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Dose range finding study:

In the dose range finding study, SANTOFLEX 14 was dosed at 1.14 ml/kg (1050 mg/kg) as received. Rats dosed with SANTOFLEX 14 did not exhibit any pharmacotoxic signs until day three. On Day 3 and 7, the animals exhibited moderate pharinacotoxic signs. At the sponsors request, two other groups of rats were dosed with higher volumes: 1.63 ml/kg (1500 mg/kg) and 2.17 ml/kg (2000 mg/kg) of SANTOFLEX 14 with the last dose being near the LD50 (2100 mg/kg). Both females dosed at 2.17 mi/kg (2000 mg/kg) died by Day 3 and both males by Day 4. Both females dosed at 1.63 mi/kg (1500 mg/kg) also were dead by Day 3. The males exhibited severe pharmacotoxic signs through Day 6 when sacrificed at the sponsors request.

Based on these results, two other groups of rats were dosed with lower volumes of 1.30 mi/kg (1200 mg/kg) and 1.2 mi/kg (1100 mg/kg). Both groups of rats exhibited from moderate to severe pharinacotoxic signs. Several females of these dose groups lost approximately 12-14 grams of body weight by the Day 2 observation. However, the males had minimum weight loss of (4-7 grams) by the Day 2 observation.

Based on these findings and with the sponsors approval, a dose level of 1100 mg/kg was chosen as the dose for evaluation in the metaphase analysis, as an estimate of the maximum tolerated dose.

Main experiment:

Clinical signs:

During the In Vivo phase of the assay, rats were observed for pharmacotoxic effects in the metaphase assay immediately after dosing and again just prior to coichicine administration.

No pharmacotoxic signs were observed in animals administered SANTOFLEX 14 immediately after dosing. Pharmacotoxic signs observed prior to colchicine administration in all groups treated with SANTOFLEX 14 were as follows:

6 Hour Group - (observed approximately 4 hours after dosing): All males had decreased body tone and piloerection. All females had decreased body tone and vocalization on touch. In addition, two females had arched back and abnormal stance.

18 Hour Group - (observed approximately 16 hours after dosing): Two males had decreased body tone. One male had vocalization when touched, abnormal stance and arched back. Another male had piloerection and diarrhea. The other two males had no apparent pharmacotoxic signs. All females had decreased body tone with four females exhibiting piloerection

and abnormal stance. Two females made vocalizations when touched. One female had also arched back.

30 Hour Group - (observed approximately 28 hours after dosing): All rats had decreased body tone and piloerection. Three males and two females made vocalizations when touched. One male and one female had an arched back. In addition, one female had abnormal gait.

These observations suggested that the rats dosed with Santoflex 14 at 1100 mg/kg exhibited moderate to severe pharmacotoxic signs indicating that these animals were dosed at or near the maximum tolerated dose.One of the males in the positive control group exhibited decreased body tone.No pharmacotoxic signs were observed in the other rats administered the vehicle or the positive controls.

Metaphase analysis:

Proportion of Cells with Aberrations:

The proportion of cells with one or more aberrations was analyzed by Chi-square analysis, by comparing the proportion of cells with aberrations in each treatment group versus the negative control. Each time of sacrifice was analyzed separately and groups within each sacrifice time were compared individually to the negative control. Cells with gaps only were not considered aberrant for this

analysis. A statistically significant increase (p 0.01) was detected with the positive control CP group at the 18 hour sacrifice. In addition, four metaphases with multiple aberrations were found in the positive control CP group. These unscorable metaphases were not included in the 100 metaphases analyzed for statistical significance. These metaphases comprised 3.85% of

the total aberrant metaphases found in that group. No other significant differences were noted.

Analysis of Aberrations per Cell:

The mean number of aberrations per cell per animal was analyzed for statistically significant increases by one-tailed t

tests for each time interval. Data reflect the total number of aberrations seen in all cells scored per group. No statistically significant increases (p 0.05) from the negative controls were detected by this analysis in animals treated with SANTOFLEX 14.

CONCLUS ION

Statistical analyses of the data indicated that SANTOFLEX 14 (1100 mg/kg) did not produce significant increases in the number of aberrations or in the number of aberrant metaphases at any of the three sacrifice times evaluated. Pharmacotoxic signs observed during the study indicated that Santoflex 14 was dosed near the maximum tolerated dose. Therefore, SANTOFLEX 14 was judged

negative in its ability to induce structural chromosomal aberrations to the hemopoietic cells of the rat bone marrow under the experimental conditions of this assay.

Proportion of cells with one or more aberrations

 Compound  dose (mg/kg)   Harvest time (h) No. of cells with one or more aberration(500 cells)  Mean proportionof aberrant cells per animal +/- SD  % aberrant cells per group
 negative control  0  6  2  0.004 ± 0.008  0.4
 Santoflex 14  1100  6  2  0.004 ± 0.008  0.4
 negative control  0  18  6  0.012 ± 0.014  1.2
 Santoflex 14  1100  18  6  0.012 ± 0.019  1.2
 CP (positive control)  20  18 251   0.502 ± 0.04**  50.2
 negative control  0  30  1  0.002 ± 0.006  0.2
 Santoflex 14  1100  30  5  0.01 ± 0.014  1.0

** significant different from negative control (p<0.01)

Applicant's summary and conclusion