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Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report date:
2004

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
OECD guideline 474, Genetic Toxicology: Mammalian Erythrocyte Micronucleus Test, adopted 21 July 1997.
Deviations:
yes
Remarks:
See "Any other information" for details
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Dioctyltin oxide
EC Number:
212-791-1
EC Name:
Dioctyltin oxide
Cas Number:
870-08-6
Molecular formula:
C16H34OSn
IUPAC Name:
dioctylstannanone
Test material form:
solid
Details on test material:
Name: dioctyloxostannane
Common name: dioctyltin oxide
Abbreviation: DOTO
Physical appearance: colourless solid
CAS number: 870-08-6
Molecular formula: C16H34OSn
Molecular weight: 316.14g.mol-1
Solubility in water: poorly soluble
Melting point: 245-260°C
Density: 1.2 g.cm-3 at 20 °C

Purity: 94.64% dioctyltin oxide
1.48% monooctyltin oxide
3.33% dioctyltin dichloride
0.53% trioctyltin oxide

Storage temperature range: < -18°C
Protected from light: yes
ORTEP Lot no.: W01/61
Expiry date: 1 October 2003
Specific details on test material used for the study:
No further details specified in the study report.

Test animals

Species:
mouse
Strain:
Swiss
Details on species / strain selection:
Species selected in accordance with test guideline
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals, housing and care
For the dose-range finding acute toxicity test ( 6 males and 6 females) and for the main micronucleus test (37 males), young adult swiss mice, were obtained from a colony maintained under SPF conditions at Charles River Deutschland, Sulzfeld, Germany. The animals arrived on 29 October 2003 (dose-range finding acute toxicity test) and 19 November 2003 (main micronucleus test).
The mice were taken in their unopened shipping containers directly to room number 5.2.15 (dose-range finding acute toxicity test and main micronucleus test). After routine serological examination, carried out on the day following arrival, the animals for the dose-range finding acute toxicity test stayed in the same room. The animals for the main micronucleus test were moved to room 5.2.08 The results of this serological examination were satisfactory. The animals were housed in sterilised Macrolon cages (type I and II), fitted with a grid cover of stainless steel and with a bedding of wood shavings (Espen E0Ol). During the quarantine and acclimatization period the animals were observed daily for overt signs of ill health and anomalies. Housing conditions were conventional. 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.

Feed and drinking water
With the exception of the fasting period prior to dosing, feed and drinking water were provided ad libitum from the arrival of the animals until the end of the study. Fresh pellet diet was provided once weekly.
The animals received a commercial rodent diet (Rat and Mouse No. 3 Breeding Diet, RM3). Batch 3142 (expiry date, 12 March 2004) was used for both the dose-range finding acute toxicity test and the main micronucleus test.
Each batch of this diet is analysed by the supplier (SDS Special Diets Services, Witham, England) for nutrients and contaminants.
The drinking water (tap-water) was given in polypropylene bottles, which were cleaned weekly and filled as needed. Tap-water for human consumption (quality guidelines according to Dutch legislation based on the EEC Council Directive 98/83/EEC), was supplied by N.V. Hydron Midden-Nederland.
Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to TNO Nutrition and Food Research. In addition, the supplier periodically (twice per year) analyses water samples taken on the premises of TNO in Zeist for a limited number of variables.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn-oil was used as vehicle for the test substance.
Details on exposure:
On the day (day-I) before the dosing day (day 0), the test substance was suspended in corn-oil, at a stock concentration of 100 mg/ml. This stock concentration was stirred overnight on a magnetic stirrer. The two lower concentrations of 50 mg/ml and 25 mg/ml were prepared prior to dosing on day 0.
Duration of treatment / exposure:
Single dose / 24-48 hour exposure
Frequency of treatment:
Single dose
Post exposure period:
No post exposure period specified
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Dose range finding: 4 animal (2 males/2females)
Main study: 5 males per dose (24 hour observation time), plus 5 males per group in control and highest dose with 48 hour observation time
Control animals:
yes, concurrent vehicle
other: Positive control
Positive control(s):
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.

Examinations

Tissues and cell types examined:
Micronuclei are small secondary nuclear structures resulting either from chromosome breakage or from malfunction of the spindle apparatus, which regulates division of the nucleus at cell division. Micronuclei can easily be visualized in erythrocytes because these cells lack a nucleus.
Details of tissue and slide preparation:
Signs of reactions to treatment were recorded from 1- 4 hours after treatment and daily thereafter. All clinical signs, observed during the performance of the study, are recorded in paragraph 3 .1 ( dose-range finding acute toxicity test) and paragraph 3.3 (main micronucleus test).
At the sacrifice time of 24 hours after dosing, 5 mice treated with the vehicle control, 15 mice treated with the test substance Dioctyloxostannane (5 mice of each 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 substance Dioctyloxostannane (2000 mg/kg-bw), were killed by cervical dislocation.
From each mouse, the bone marrow cells of both femurs were immediately collected into foetal calf serum and processed into glassdrawn smears according to the method described by Schmid (1976). Two bone marrow smears per animal were prepared, air-dried and fixed in methanol. One smear per animal was stained with a May-Grtinwald Giemsa solution. The other smear was stored as 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.
Evaluation criteria:
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 substance 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.
A test substance 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 substance or its metabolites are considered to have reached the general circulation and thereby the bone marrow, if the test substance 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:
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 homogeneous, separate variance t-tests. These t-tests were applied to the negative control group A versus treatment groups B, C and D. Furthermore, the positive control group E and the negative control group A were compared using pooled error variance t-tests or, if variances were not homogeneous, separate t-tests. In addition a linear trend test (orthogonal contrasts) was applied across groups A, B, C and D.
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 homogeneous, 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).

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.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Dose-range finding acute toxicity test
Due to a failed oral administration, female 3 died on the day following administration. No severe clinical signs as a result of the test substance Dioctyloxostannane, or sex differences, were observed in the dose-range finding acute toxicity test. 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 same three dose levels of the test substance Dioctyloxostannane (2000, 1000 and 500 mg/kg-bw), administered in the dose-range finding acute toxicity test and with male mice only.

Clinical signs in the main micronucleus test
At 24 hours after administration, all mice of the C group (Dioctyloxostannane; 1000 mg/kg-bw) and D group (Dioctyloxostannane; 2000 mg/kg-bw) showed a hunched posture, piloerection and a wet anal area as a result of diarrhoea caused by the test substance.
At 48 hours, mouse D78 (Dioctyloxostannane; 2000 mg/kg-bw) still showed a hunched posture, piloerection and a wet anal area as a result of diarrhoea.

Statistical analysis of the main micronucleus test results
At both sacrifice times of 24 hours and 48 hours after treatment, the two-way ANOVA did not yield a statistically significant effect for MPE and PE. This indicates that treatment with Dioctyloxostannane, up to 2000 mg/kg-bw (the limit dose level), did not result in genotoxicity or clastogenicity to the bone marrow target cells.
At the sacrifice time of 24 hours, in the positive control group, the incidence of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) was statistically significantly different (P<0.001) from the negative control A. This demonstrates the validity of the test system.
The results of this micronucleus test did not show any indication of chromosomal damage and/or damage to the mitotic apparatus of the bone marrow target cells in male mice, treated orally with the test substance Dioctyloxostannane.

Any other information on results incl. tables

Dose-range finding acute toxicity test

Clinical signs during the dose-range finding acute toxicity test

Test substance: Dioctyloxostannane

Vehicle: corn-oil

Route: by gavage

Dosing volume: 20 ml/kg-bw

Test substance and dose level (mg/kg-bw)

1h after1)administration

4 h after1)administration

24 h after1)administration

48 h after1)administration

Animal number and sex

Dioctyloxostannane 500 mg/kg-bw

 

sl; se; br

sl; se; p; br; †

 

Male 2

Male 4

Female 1

Female 3

Dioctyloxostannane 1000 mg/kg-bw

 

 

 

 

Male 6

Male 8

Female 5

Female 7

Dioctyloxostannane 2000 mg/kg-bw

 

 

 

 

Male 10

Male 12

Female 9

Female 11

1) empty cells, no clinical signs observed

sl: sluggishness

se: slit-eyes

p: piloerection

br: irregular breathing

†: animal died

 

Body weight in the main micronucleus test

Body weights of the animals prior to the start of treatment

Treatment group

Dose level (mg/kg-bw)

Sex

N

Bw mean (g)*

Bw SEM (g)

A / negative control (corn-oil)

0

M

10

34.83

0.56

B / Dioctyloxostannane

500

M

5

34.38

0.43

C / Dioctyloxostannane

1000

M

5

34.12

0.49

D / Dioctyloxostannane

2000

M

10

34.22

0.55

E / Mitomycin C# (positive control)

0.75

M

5

33.70

0.36

#: Sigma; single (10 ml/kg-bw) intraperitoneal injection (vehicle: saline)

M: male

*: individual body weights are stored in the archives

 

Micronucleated Polychromatic Erythrocytes (MPE) in the mice of the main micronucleus test

The group mean numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE)

Group:

A

Neg. contr. (corn-oil)

Dioctyloxostannane

(dose level in mg/kg-bw)

E

pos. control mitomycin C (0.75 mg/kg-bw)

Sex

† (h)

B

500

C

1000

D

2000

Male

24

2.0 ± 0.7

2.6 ± 1.1

1.8 ± 0.4

1.4 ± 0.5

50.4 ± 10.3***

Male

48

2.2 ± 1.1

-

-

1.4 ± 0.9

-

Means and standard deviations: ***P<0.001 (t-test)l group size: 5

The positive control group E, at time point 24h, differed significantly from the negative control A (P<0.01).

 

Polychromatic Erythrocytes (MPE) in the mice of the main micronucleus test

Group mean numbers of polychromatic erythrocytes (MPE) per 2000 erythrocytes (E)

Group:

A

Neg. contr. (corn-oil)

Dioctyloxostannane

(dose level in mg/kg-bw)

E

pos. control mitomycin C (0.75 mg/kg-bw)

Sex

† (h)

B

500

C

1000

D

2000

Male

24

89.2 ± 15.2

85.2 ± 14.3

91.0 ± 14.4

92.0 ± 18.2

77.0 ± 7.6

Male

48

88.0 ± 10.8

-

-

75.2 ± 5.2

-

 

Historical data for the Micronucleus Test in Swiss Mice (CD-1 strain)

 

Historical negative controls

Historical negative control data (vehicle, 1 or 2 treatment) from 30 studies carried out from 1999-2003. Overview from 20 May 2003.

Vehicle

Route

Micronucleated PE per 1000 PE at 24-72 hours post-treatment, males and females

Mean ± standard deviation

Range of means

Saline, water or PBS#

 

 

Corn-oil

Orla

i.p.

i.v.

 

oral

1.9 ± 1.0

2.1 ± 1.3

2.1 ± 1.0

 

2.6 ± 1.3

1.6-2.2

1.6-2.6

1.2-3.2

 

1.0-4.8

# PBS = phosphate-buffered saline

 

Historical positive controls

Historical positive control data from studies carried out from 1999-2003. Overview from 20 May 2003.

Vehicle

Route

Micronucleated PE per 1000 -2000 PE at 24 hours post-treatment, males and females

Mean ± standard deviation

Range of means

Mitomycin C 1.5 mg/kg-bw

 

Mitomycin C 0.75 mg/kg-bw

i.p.

 

i.p.

49 ± 17

 

36 ± 9

17-95

 

17-70

 

Applicant's summary and conclusion

Conclusions:
The data supports the conclusion that, under the conditions used in this study, the test substance Dioctyloxostannane did not produce chromosomal damage or damage to the mitotic spindle apparatus in the bone marrow target cells of mice.
Executive summary:

The test substance Dioctyloxostannane (DOTO) [CAS# 870-08-6] 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 and a main micronucleus test with male mice only.

 

Results of the dose range finding acute toxicity test indicated that there were no sex differences in response and that a limit dose of 2000 mg/kg-bw could be tolerated. Male mice were chosen for the main study and doses of 2000, 1000 and 500 mg/kg-bw were adopted.

 

For the main micronucleus test, animals were treated once by gavage with three graded dose levels of the test substance Dioctyloxostannane. The high dose group (D) consisted of 10 males and each animal received a dose of 2000 mg/kg-bw (the limit dose-level). The moderate dose group (C) consisted of 5 males and each animal received a dose of 1000 mg/kg-bw. The low dose group (B) consisted of 5 males and each animal received a dose of 500 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 substance, 5 negative control animals and 5 positive control animals, were euthanized. At 48 hours after treatment, the remaining 5 animals of group D (the high dose group) and the remaining 5 negative control animals, were euthanized. 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 time points, 24 and 48 hours after treatment, the number of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) were counted for each mouse. The mean number of MPE per 2000 PE, at dose levels of 2000, 1000 and 500 mg/kg-bw of Dioctyloxostannane, were not statistically significantly different from the vehicle control mean. Therefore, the test substance Dioctyloxostannane, at dose levels up to 2000 mg/kg-bw, was not genotoxic to bone marrow cells in mice.

 

For the mice of the positive control group, the mean number of MPE per 2000 PE was significantly (p<0.001) elevated compared to the mean of the vehicle control mice. This demonstrates the validity of the test system.

 

At 24 and 48 hours after treatment, the mean number of polychromatic erythrocytes (PE) per erythrocyte (E) in mice, at all treatment levels of Dioctyloxostannane, were not statistically significantly different from the mean of the vehicle control mice. Therefore, treatment with Dioctyloxostannane, at dose-levels up to 2000 mg/kg-bw, was not cytotoxic to the bone marrow of mice.

 

The data supports the conclusion that, under the conditions used in this study, the test substance Dioctyloxostannane did not produce chromosomal damage or damage to the mitotic spindle apparatus in the bone marrow target cells of mice.