Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP. Preferred study for this SIDS endpoint.

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:
The results of the total tin analysis indicated that the amount of total tin in the bone marrow samples was less than the detection limit of 250 ng. The main test was performed without total tin analysis. Deviation did not adversely influence the integ
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Trichloromethylstannane
- Molecular formula (if other than submission substance): CH3Cl3Sn
- Molecular weight (if other than submission substance): 240.8 g/mol
- Smiles notation (if other than submission substance): CL[Sn](CL)(CL)C
- Structural formula attached as image file (if other than submission substance): see Fig.
- Physical state: liquid
- Analytical purity: 98.53%
- Composition of test material, percentage of components: 98.53% trichloromethylstannane:1.32% dichlorodimethylstannane: 0.15% tetrachlorostannane
- Lot/batch No.: W 2001/47
- Expiration date of the lot/batch: July 31, 2003
- Storage condition of test material: at -18+/-2 degrees C, in the absence of light

Test animals

Species:
rat
Strain:
other: Wistar outbred CrI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: young adult
- Weight at study initiation: 162.6 to 203.4
- Assigned to test groups randomly: yes, by computer randomization
- Fasting period before study: yes, 19 hours prior to dosing
- Housing: sterilized Macrolon cages (type IV for the main test and type III for the dose-range finding test) fitted with a grid cover of stainless steel
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 degrees C
- Humidity (%): at least 30% not exceeding 70% except during room cleaning
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark


IN-LIFE DATES: From: October 1, 2002 To: October 3, 2002

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.9% sodium chloride
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle: 50, 16.67, 5.56, and 1.85 mg/ml
- Amount of vehicle (if gavage or dermal): 20 ml/kg-bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: On the day before dosing the test substance was weighed and stored. On day 0, the test substance was dissolved and diluted in 0.9% sodium chloride at concentrations of 50, 16.67, 5.56 and 1.85 mg/ml. The orally (by gavage) given dosing volume was 20 ml/kg-bw.


DIET PREPARATION N/A- gavage administration
Duration of treatment / exposure:
single dose
Frequency of treatment:
single dose
Post exposure period:
24 hours for all doses including controls and 48 hours for the negative control and two highest dose levels
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 37, 111, 333, 1000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Group A, D, E: 10 males per group. Group B, C,  F: 5 males per group.
Control animals:
yes, concurrent vehicle
other: positive control- Mitomycin C
Positive control(s):
mitomycin C
- Justification for choice of positive control(s): no data
- Route of administration: intraperitoneal injection; vehicle: saline
- Doses / concentrations: 10 ml/kg-bw

Examinations

Tissues and cell types examined:
- Clinical observations: Clinical signs were recorded from 1-4 hours and  at 24 and 48 hrs after treatment. - Organs examined at necropsy: From each rat, the bone marrow cells of  one femur were immediately collected into foetal calf serum and processed  into glassdrawn smears according to the method described by Schmid  (1976).  No  additional organs were examined.
Details of tissue and slide preparation:

OTHER: From each rat, the bone marrow cells of one femur were immediately collected into fetal 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-Grünwald  Giemsa solution. The second fixed smear was stored as a reserve slide.  Slides were independently coded.
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.
Evaluation criteria:
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/2000 PE was registered, and the number  of MNE/NE. 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 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:
STATISTICAL METHODS: 1) At time point 24 hours after administration, data  on micronucleated polychromatic erythrocytes (MPE) and polychromatic  erythrocytes (PE) were subjected to a One Way Anova with factor group (A,  B, C, D and E). If the Anova yielded a significant effect (p<0.05), it  was followed by pooled error variance t-tests or, if variances were  inhomogenous, separate variance t-tests. These t-tests were applied to  the negative control group A versus treatment groups B (37 mg/kg bw), C  (111 mg/kg bw), D (333 mg/kg bw) and E (1000 mg/kg bw). In addition, the  positive control group F and the negative control group A, at time point  24 hours after administration, were compared using t-tests and a linear  trend test across groups A, B, C, D and E (orthogonal contrasts). 2) At time point 48 hours after administration, data on MPE and PE in  group A, D and E were subjected to a One Way Anova. If the Anova yielded  a significant effect (p<0.05), it was followed by pooled error variance  t-tests or, if variances were inhomogenous, separate variance t-tests. In  addition a linear trend test across groups A, D and E were applied  (orthogonal contrasts).

Results and discussion

Test results
Sex:
male
Genotoxicity:
positive
Remarks:
weakly genotoxic because of a potential to produce micronuclei and induce damage to the mitotic spindle apparatus in the bone marrow target cells.
Toxicity:
no effects
Remarks:
MORTALITY: None. CLINICAL SIGNS: None. No treatment related cytotoxicity could be demonstrated in the target cells of the rat bone marrow.
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 25, 50, 100  and 200 mg/kg bw
- Clinical signs of toxicity in test animals: No clinical signs were observed at any of the dose levels.
- Rationale for exposure: performed in order to determine the dose level(s) to be used in the main micronucleus test.
- Harvest times: On day 2 (48 hours after administration), the negative control rats (2  males and 2 females) and the rats (2 males and 2 females) of the highest  dose-level (200 mg/kg bw) were killed and the bone marrow cells of two  femurs of each rat were sampled in 0.9 % sodium chloride for total tin  analysis with the atomic absorption spectrometry with graphite furnace  technique (GF-AAS) at a wavelength of 224.6 nm
- High dose with and without activation: The  limit dose of 2000 mg/kg bw could not be administered because the test substance is known to be harmful to the rats and the acute toxicity  (LD50) is between 200 and 2000 mg/kg bw (data provided by the sponsor).  Therefore, 200 mg/kg bw was selected as highest dose level for the  dose-range finding acute toxicity test.
- Other: The results of these  analyses indicated that the amount of total tin in the bone marrow  samples was less than the detection limit of 250 ng. In agreement with  the sponsor, it was decided to continue the study with a main micronucleus test excluding total tin analysis in bone marrow samples. No sex differences were observed in the dose range-finding study;  therefore, the main study was performed with male rats only.


RESULTS OF DEFINITIVE STUDY EFFECT ON PCE/NCE RATIO: 24 hours after treatment, measured as the mean  number of MPE, the negative vehicle control group A (0.9 % sodium chloride) was statistically significantly (weak effect) different from treatment group B (test substance; 37 mg/kg bw; *p<0.05), treatment group  D (test substance; 333 mg/kg bw; *p<0.05) and treatment group E (test  substance; 1000 mg/kg bw; *p<0.05). In group C (test substance; 111 mg/kg  bw), the mean number of MPE was not statistically significantly higher  than the mean number of MPE found in the vehicle control group A.  The  linear trend test showed a weak significant effect (*p<0.05). 48 hours  after treatment no statistical differences between the negative control  group and the treated groups could be demonstrated. At both time points of 24 and 48 hours after treatment, the numbers of  polychromatic erythrocytes (PE) per number of (E) erythrocytes in the  rats treated with four different dose levels (37, 111, 333 and 1000 mg/kg  bw) of the test substance Trichloromethylstannane, were not statistically  significantly different from the numbers of polychromatic erythrocytes  per number of erythrocytes found in the vehicle controls. 
- Induction of micronuclei (for Micronucleus assay): weakly genotoxic because  of a potential to produce micronuclei and induce damage to the mitotic  spindle apparatus in the bone marrow target cells. LOAEL (C): The lowest concentration at which a weak genotoxic effect was  observed, was 37 mg/kg bw. 
- MPE/2000 PE FREQUENCY: After 24 hrs the observed MPE/2000 PE frequencies  were: group A (control), 1.2+/-0.4; group B (37 mg/kg bw), 3.0+/-1.2;  group C (111 mg/kg bw), 1.8+/-0.4; group D (333 mg/kg bw), 3.0+/-1.4;  group E (1000 mg/kg bw), 3.4+/-1.7; group F (positive control),  26.8+/-3.3. After 48 hrs the observed frequencies were: group A (negative  control), 2.4+/-1.8; group D (333 mg/kg bw), 1.8+/-1.1; group E (1000  mg/kg bw), 1.6+/-0.9.
- Statistical evaluation:At the 24-hour time point, the PCE/NCE ratio in  group B, D and E was significantly higher than for the negative control  group, p<0.05. The difference in PCE/NCE ratio was not statistically  significant between the negative control group and group C at the 24-hour  and 48-hour time points, and the 48-hour time points for group B, D and E. The numbers of polychromatic erythrocytes (PE) per number of (E)  erythrocytes were not statistically significantly different from the  numbers of polychromatic erythrocytes per number of erythrocytes found in  the vehicle controls for any of the dose groups or time points.

Any other information on results incl. tables

MORTALITY: None.
CLINICAL SIGNS: None.

No treatment related cytotoxicity could be demonstrated in the target cells  of the rat bone marrow.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): positive
From the results obtained, under the conditions used, it was demonstrated that the test substance was weakly genotoxic because of a potential to produce micronuclei and induce damage to the mitotic spindle apparatus in the bone marrow target cells
Executive summary:

The test substance was examined in a bone marrow micronucleus test in rats. The study consisted of a dose-range finding acute toxicity test and a main micronucleus test.

In the dose range finding acute toxicity test, bone marrow cells of 2 males and 2 females, treated with the highest dose level of the test substance (200 mg/kg-bw), was sampled for total tin analysis. The results of this analysis were not satisfactory because the amount of total tin in the bone marrow samples was less than the detection limit. Therefore, the main micronucleus test was performed without total tin analysis in bone marrow samples.

For the main micronucleus test, animals were treated once by gavage with four graded dose levels of the test substance. ne group, consisting of 10 males, was treated with a dose level of 1000 mg/kg. One group, consisting of 10 males, was treated with a dose level of 333 mg/kg. One group, consisting of 5 males, was treated with a dose level of 111 mg/kg. One group, consisting of 5 males, was treated with a dose level of 37 mg/kg. The rats of the vehicle control group were treated in a similar way with the vehicle only (saline; 0.9% sodium chloride). A positive control group (5 males) was given a single intraperitoneal dose of the mutagen mitomycin C. At time point 24 hours after treatment, 5 animals of each dose level of the test substance, 5 negative control animals and 5 positive control animals, were sacrificed. At time point 48 hours after treatment, 5 animals of the two highest dose levels of the test substance together with 5 negative control animals were sacrificed. From one femur of each animal, the bone marrow cells were collected in fetal calf serum and processed into smears for microscopic examination.

At time point 24 hours after treatment, the mean numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE) in rats, treated with three different dose levels (37, 333 and 1000 mg/kg) of the test substance, were weakly statistically significantly higher than the mean number found in the vehicle control rats. The linear trend test also yielded a weak significant effect. Therefore, the study indicates that treatment with the test substance at dose levels up to 1000 mg/kg, resulted in a weak genotoxic effect to bone marrow cells in rats. The mean number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in rats, treated with a dose level of 111 mg/kg, was not statistically significantly higher than the mean number found in the vehicle control rats. For the rats of the positive control group, the mean number of MPE per 2000 PE was statistically significantly higher than the mean number found in the vehicle control rats. This deomstrates the validity and sensitivity of the test system.

At time point 48 hours after treatment, the mean numbers of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in rats, treated with the two highest dose levels (333 and 1000 mg/kg) of the tet substance, were not statistically significantly higher than the mean number found in the vehicle control rats.

At both time points of 24 hours and 48 hours after treatment, the numbers of polychromatic erythrocytes per number of erythrocytes in rats, treated with four different dose-levels (1000, 333, 111 and 37 mg/kg) of the test substance, were not statistically significantly different from the polychromatic erythrocytes per number of erythrocytes found in the vehicle control rats. Therefore, no treatment related cytotoxicity to bone marrow cells of male rats could be demonstrated in the target cells.

From the results obtained in this micronucleus test with the test substance, it was demonstrated that the test substance was weakly genotoxic to bone marrow cells of rats and that the test substance has the potential to induce damage to the mitotic spindle apparatus of the bone marrow target cells.