Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 January 2003 to 28 April 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
See principles of method if other than guideline
Principles of method if other than guideline:
Deviations from the protocol:
- The dose range finding toxicity test was started on 20 December 2002, instead of 29 October 2002.
- The main micronucleus test was started on 14 January 2003, instead of 26 November 2002.
- During the main micronucleus test, during a short period, the relative humidity in animal room 5.2.10 was slightly higher than 70 %, due to cleaning activities.
- During the main micronucleus test, during a short period, the pressure in animal room 5.2.10 was slightly lower than 5 %, due to cleaning activities.

These deviations did not adversely influence the integrity and/or the validity of the study.
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: a transparent colourless to pale yellow liquid
- Storage condition of test material: at <-18 °C, in the absence of light


Test animals

Species:
mouse
Strain:
Swiss
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: young adult
- Weight at study initiation: Approx 31-35 g
- Assigned to test groups randomly: Yes, under following basis: animals were allocated by computer randomization to a vehicle control group A, three test groups treated with Tributylchlorostannane (B, C and D) and to a positive control group E.
- Fasting period before study: Administration of the test substance was carried out after a fasting period of 2 hours and 15 minutes.
- Housing: The animals were housed in sterilised Macrolon cages (type I + II), fitted with a grid cover of stainless steel and with a bedding of sterilised softwood chips.
- Diet: With the exception of the fasting period prior to dosing, feed was provided ad libitum from the arrival of the animals until the end of the study.
- Water: With the exception of the fasting period prior to dosing, drinking water was provided ad libitum from the arrival of the animals until the end of the study. The drinking water (tap-water) was given in polypropylene bottles, which were cleaned weekly and filled as needed.
- Acclimation period: During the quarantine and acclimatization period (at least 6 days) the animals were observed daily for overt signs of ill health and anomalies.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: Relative humidity of at least 30 % and not exceeding 70 % other than during room cleaning.
- Air changes (per hr): The animal rooms were ventilated with about 10 air changes per hour
- Photoperiod (hrs dark / hrs light): Lighting was artificial with a sequence of 12 hours light and 12 hours dark.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On day 0 (prior to dosing), the test material was suspended in corn-oil, at concentrations of 15, 7.5 and 3.75 mg/mL. The orally (by gavage) given dosing volume was 20 mL/kg-bw. Prior to dosing, the animals were weighed. Administration of the test material was carried out after a fasting period of 2 hours and 15 minutes. The dose-levels administered were 300, 150 and 75 mg/kg-bw.
Duration of treatment / exposure:
Single oral dose
Frequency of treatment:
Single oral dose
Post exposure period:
48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals for doses 75, 150 mg/kg-bw and the positive control group. 10 animals used for the negative control and dose group 300 mg/kg-bw. Three reserve mice were treated with the highest dose-level of 300 mg/kg-bw to replace any mortality in the highest dose-level group.
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Route of administration: Sigma; intraperitoneal injection
- Vehicle: saline
- Doses / concentrations: stock-concentration: 0.075 mg/ml; dosing volume 10 ml/kg-bw; dose level: 0.75 mg/kg-bw
- Other: For safety reasons, the animals of the positive control group (main micronucleus test only) were housed in a laminar down-flow cabinet, just prior to administration and until sacrifice. The animal rooms were ventilated with about 10 air changes per hour and were maintained at a temperature of 22 ± 3 °C and a relative humidity of at least 30 % and not exceeding 70 % other than during room cleaning. Lighting was artificial with a sequence of 12 hours light and 12 hours dark.

Examinations

Tissues and cell types examined:
Bone marrow cells.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The acute toxicity (LD50) for rats, orally treated with the test material was not known. Therefore, dose levels of 2000 (the limit dose), 750, 375 and 187.5 mg/kg-bw were selected for the dose-range finding acute toxicity test in male and female mice.

The dose levels and the choice of sex, selected for the main micronucleus test, were based on the clinical signs observed during the performance of the dose-range finding acute toxicity test as described below. For both sexes, severe toxicity was observed in this dose-range finding acute toxicity test at the three highest dose levels of 2000, 750 and 375 mg/kg-bw. These results obtained were discussed with the sponsor. Thereafter, it was decided to perform the main micronucleus test with dose levels of 300, 150 and 75 mg/kg-bw and with male mice only.

Dose-range finding acute toxicity test:
A dose-range finding acute toxicity test was performed in order to determine the dose level(s) and sex to be used in the main micronucleus test. Dose levels of 2000 (the limit dose), 750, 375 and 187.5 mg/kg-bw were selected for the dose-range finding acute toxicity test in male and female mice.
After a fasting period of ca. 2 hours and 45 minutes, two males and two females were treated once (by gavage) with each of the four dose-levels (2000, 750, 375 and 187.5 mg/kg-bw) of the test material. Observations with respect to all signs of reaction to treatment were recorded 1 hour, 4 hours, 1 day and 2 days post treatment. The animals were coded by an earmark (V-mark in the ears). The females were allocated odd numbers and the males even numbers. Body weights were recorded prior to dosing (day 0) and on day 3. Sex differences could not be demonstrated in the dose-range finding acute toxicity test.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Signs of reactions to treatment were recorded from 1- 4 hours after treatment and daily thereafter.

At the sacrifice time of 24 hours after dosing, 5 mice treated with the vehicle control, 15 mice treated with the test material (5 mice per dose-level) and 5 mice treated with the positive control substance mitomycin C, were killed by cervical dislocation. At the sacrifice time of 48 hours after dosing, 5 mice treated with the vehicle control, together with 5 mice treated with the highest dose-level of the test material, were killed by cervical dislocation.


DETAILS OF SLIDE PREPARATION:
From each mouse, the bone marrow cells of both femur were immediately collected into foetal calf serum and processed into glassdrawn smears. Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grunwald Giemsa solution. The other smear was stored as a reserve slide.

The slides were randomly coded by a person not involved in the scoring of slides. The slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines taking care that areas selected for evaluation were evenly distributed over the whole smear.
The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 200 erythrocytes (E) per animal; if micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 200 E (PE + NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. Thus the incidence of MPE was recorded in a total of 2000 PE per animal and the number of MNE was recorded in the number of NE.

Due to the unexplained reduction in MPE in the 48 hour control group, the sponsor requested that the slides of the 48 hours vehicle control animals be recounted to verify the results of the first count.

Evaluation criteria:
The study is considered valid if the positive controls give a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls are within the historical range.

A response is considered to be positive if the mean number of MPE/2000 PE is statistically significantly higher, when compared to the mean number of the vehicle controls.

A test material is considered to cause chromosomal damage and/or damage to the mitotic apparatus, if a clear dose-related increase in the mean numbers of MPE/2000 PE is observed, when compared to the mean number of the vehicle controls, and/or if a single positive dose level is observed.

A test material is considered to be negative in the micronucleus test if it produces no positive response at any of the dose-levels and time points analysed.

The test material or its metabolites are considered to have reached the general circulation and thereby the bone marrow, if the test material statistically reduce the mean number of PE/E or causes systemic toxicity.

Both statistical significance and biological relevance are considered together in the evaluation.
Statistics:
1) At time point 24 hours after administration, data on MPE and PE were subjected to a One Way Anova with factor group (A,B,C and D). If the Anova yielded a significant effect (p<0.05), it was followed by pooled error variance t-tests or, if variances were not homogenous, separate variance t-tests. These t-tests were applied to the negative control group A versus treatment groups B, C and D. In addition, the positive control group E and the negative control group A were compared using pooled error variance t-tests or, if variances were not homogenous, separate t-tests.

2) At time point 48 hours after administration, for treatment groups A and D, data on MPE and PE were subjected to pooled error variance t-tests or, if variances were not homogenous, separate variance t-tests.

All statistical tests were performed using BMDP statistical software (W.J. Dixon, BMDP Statistical Software Manual, University of California Press, Berkeley, 1992).

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
(Equivocal)
Toxicity:
yes
Remarks:
300 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Severe clinical signs were observed in the dose-range finding acute toxicity test. These severe clinical signs were observed in both male and female mice. The results of the dose-range finding acute toxicity test were reported to the sponsor. Thereafter, it was decided to perform the main micronucleus test with the dose levels of 300, 150 and 75 mg/kg-bw and with male mice only.

Any other information on results incl. tables

All results tables can be seen in the attached illustration.

Clinical signs in the main micronucleus test:

A-group (corn-oil): no clinical signs were observed.

B-group (75 mg/kg-bw): no clinical signs were observed.

C-group (150 mg/kg-bw): C44 showed piloerection. C46 showed blepharospasm.

D-group (300 mg/kg-bw): D62, D64, D66 and D80 showed sluggishness, blepharospasm and piloerection. D68, D70 and D74 showed sluggishness and blepharospasm. D72 died before sacrifice time and was replaced by reserve mouse D94. D76 died before sacrifice time and was replaced by reserve mouse D96 D78 died before sacrifice time and could not be replaced because one of the reserve animals died after treatment.

Micronucleated Polychromatic Erythrocytes (MPE):

In this test the incidences of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) in treatment groups B (75 mg/kg-bw) and C (150 mg/kg-bw) and D (300 mg/kg-bw) were 2.2, 2.2, and 4.0, respectively. The incidence of MPE per 2000 PE in the positive control group (E), at the 24 hour time point, was 45.0.

At the 48 hour time point there was an unexplained reduction in the incidence of MPE in the negative controls compared to the 24 hour time point. The slides were re-counted and both results are presented. At 48 hours the 4.0 incidence of MPE per 2000 PE in treatment group D was the same as the incidence at 24 hours. Also in the 300 mg/kg-bw group (D), 3 animals died prior to scheduled sacrifice at 48 hours and only 2 were able to be replaced with reserve animals leaving n=4 for this group.

Polychromatic Erythrocytes (PE) in the mice of the main micronucleus test:

At 24 and 48 hours after treatment, in the vehicle controls, the number of PE per 200 E were approximately equal. At 48 hours after treatment, the number of PE per 200 E were reduced in treatment group D (300 mg/kg-bw) relative to controls. This is a biologically relevant effect and indicates that the test substance reached the bone marrow and induced cytotoxicity to the bone marrow cells.

Statistical analysis of the main micronucleus test results:

At 24 hours after treatment, the two-way ANOVA indicated there were no statistically significant differences in the incidences of MPE and PE in any treatment group compared to the negative controls. At 24 hours, the incidence of MPE per 2000 PE in the positive control group was statistically significantly greater than (p<0.001) that in the negative controls. The latter result demonstrates the sensitivity of the test system and the validity of the assessments.

At 48 hours after treatment, the two-way ANOVA indicated a statistically significant difference between treatment group D (300 mg/kg-bw) and the negative control group for MPE (p<0.05).

At 48 hours after treatment, the ANOVA indicated a statistically significant difference between treatment group D (300 mg/kg-bw) and the negative control group for MPE (p<0.05).

This difference was judged to be due to the incidental drop in the control incidence of MPE rather than an increase in the incidence of MPE in the treated group. Two observations support this judgement: 1) the incidence data for PE per 200 E in controls at the 24 and 48 hour time points are essentially the same, and 2) the maximal number of MPE per PE among the mice in the 300 mg/kg-bw dose group is no higher than the highest incidence in mice from the vehicle controls. Thus, the observations suggest that the statistical difference, while real, is not indicative of an underlying increase in the effect of Tributylchlorostannane on MPE in this case. At 48 hours after treatment, the two-way ANOVA indicated there was a statistically significant decrease in the number of PE in treatment group D (300 mg/kg-bw) (**p<0.01), when compared to the negative control group A (corn-oil). This indicated that the test material reached the bone marrow and was cytotoxic to bone marrow cells.

Applicant's summary and conclusion

Conclusions:
Under the conditions used in this study, the test material, was cytotoxic to the bone marrow of mice. The positive control goup responded appropriately. The sole statistical difference in the incidences of MPE, specifically in the 300 mg/kg group at the 48 hour time point, was judged incidental because of the lethality seen at this dose level and the unexplained decrease in MPE in the concurrent controls. Thus, the weight of the evidence suggests the test material is not mutagenic in this test, but the data are equivocal.
Executive summary:

The test material was examined for its mutagenic potential in a bone marrow micronucleus test in mice. The study consisted of a dose-range finding acute toxicity test carried out with male and female mice followed by a main micronucleus test with male mice.

Results of the dose range finding acute toxicity test indicated that there were no differences in the response which could be attributed to the sex of the animals. Severe toxicity was observed in both sexes at dose levels of 2000, 750, and 375 mg/kg-bw. Male mice were chosen for the main micronucleus test and doses of 300, 150, and 75 mg/kg-bw were adopted.

For the main micronucleus test, animals were treated once by gavage with three graded dose levels of the test material. The high dose group (D), consisted of 10 males, and each animal received a dose of 300 mg/kg-bw. The mid dose group (C), consisted of 5 males, and each animal received a dose of 150 mg/kg-bw. The low dose group (B), consisted of 5 males, and each animal received a dose of 75 mg/kg-bw. The vehicle control group A consisted of 10 males, and each animal was dosed in a similar way with the corn-oil vehicle only. A positive control group consisted of 5 males, and each animal was given a single intraperitoneal dose of mitomycin C at 0.75 mg/kg-bw. At 24 hours after treatment, 5 animals of each dose-level of the test material, 5 negative control animals and 5 positive control animals, were euthanised. At 48 hours after treatment, the remaining 5 animals of group D, the high dose group and the remaining 5 negative control animals, were euthanised. From both femurs of each animal, the bone marrow cells were collected in foetal calf serum and processed into smears for microscopic examination. At both 24 and 48 hours after treatment, the number of polychromatic erythrocytes (PE) per 200 erythrocytes (E) and the number of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) were counted for each mouse. The group mean numbers of MPE per 2000 PE and the group mean numbers of PE per 200 E for each group were reported. The historical vehicle control and historical positive control data for MPE per 1000 PE were also presented.

At 24 and 48 hours after treatment in the vehicle controls, the number of PE per 200 E were approximately equal. At 48 hours after treatment, the number of PE per 200 E were reduced in treatment group D (300 mg/kg-bw) relative to controls. This is a biologically relevant effect and indicated that the test material reached the bone marrow and induced cytotoxicity to the bone marrow cells. At 24 hours after treatment, there were no statistically significant differences in the incidences of MPE or PE in any treatment group compared to the negative controls. At 24 and 48 hours, the incidence of MPE per 2000 PE in the positive control group was increased (P<0.001) relative to the negative controls. The latter results demonstrates the sensitivity of the test system and the validity of the assessments.

At the 48 hour time point there was an unexplained reduction in the incidence of MPE per 2000 PE in the negative controls compared to the 24 hour time point. This was confirmed in a recount of the slides. The reduced incidence of MPE per 2000 PE in the high dose group (D) at 48 hours was unchanged compared to the incidence at 24 hours. Also in the 300 mg/kg-bw group (D), 3 animals died prior to scheduled sacrifice at 48 hours and only 2 were able to be replaced with reserve animals leaving n=4 for this group. 5.At 48 hours after treatment, the ANOVA indicated a statistically significant difference between treatment group D (300 mg/kg-bw) and the negative control group for MPE (p<0.05). This difference was judged to be due to the incidental drop in the control incidence of MPE rather than an increase in the incidence of MPE in the treated group. Two observations support this judgement: 1) the incidence data for PE per 200 E in controls at the 24 and 48 hour time points are essentially the same, and 2) the maximal number of MPE per PE among the mice in the 300 mg/kg-bw dose group is no higher than the highest incidence in mice from the vehicle controls. Thus, the observations suggest that the statistical difference, while real, is not indicative of an underlying increase in the effect of Tributylchlorostannane on MPE in this case.

Under the conditions used in this study, the test material was cytotoxic to the bone marrow of mice. The positive control group responded appropriately. The sole statistical difference in the incidences of MPE, specifically in the 300 mg/kg-bw group at the 48 hour time point, was judged incidental because of the lethality seen at this dose level and the unexplained decrease in MPE in the concurrent controls. Thus, the weight of the evidence suggests the test material is not mutagenic in this test, but the data are equivocal.