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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: genome mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted with a similar substance according to standard methods and GLP, therefore it is considered reliable and relevant, but in isolation, not adequate for classification. Read-Across

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Bio Test s.r.o., Pod Zámkem 279, Konárovice, Crech Republic
- Age at study initiation: 8-10 weeks on delivery
- Weight at study initiation: Males 250 g, Females 170 g
- Assigned to test groups randomly: Yes, under following basis: the animals were sorted according to the body weight and randomly allocated to the dose group taking animals from each range group.
- Fasting period before study: Not provided
- Housing: 2-3 animals of the same sex/cage in Macrolon 2000P cages (Tecniplast), Tier Wohl (JRS, Germany) was used as bedding
- Diet (e.g. ad libitum): Standard pelletized rodent diet of monitored quality Feed NOE H4 (Racio Breclav, CZ);Feed containers were changed and sanitized at least once a week
- Water (e.g. ad libitum): Water of monitored quality, ad libitumWater containers were changed and sanitized at least once a week
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: methylcellulose 0.5%
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Each animal in treated groups received the appropriate dose of Test item suspension orally by gavage in the volume of 1 mL per 100 g body weight. The animals in the negative control group received the appropriate volume of saline solution by the same way. The animals in the positive control group were administered cyclophosphamide solution at the dose of 20 mg/kg by a single intraperitoneal application in the volume of 1 mL per 100 g body weight.

The following procedure was adopted for the preparation of a homogenous suspension: at first the weighed amount of the respective substance was ground in a grinding mortar with a small volume of carrier liquid. Then the content of the mortar was transferred into a calibrated vessel. Another amount of carrier liquid was poured into the mortar, stirred and the mortar with the pestle was rinsed, the rinsing liquid was added into the same calibrated vessel. This rinsing procedure was repeated once more. Then the volume in the calibrated vessel was made up with the carrier liquid to the required volume and mixed. Immediately before the application, the suspension was again stirred by means of an electromagnetic stirrer.


Duration of treatment / exposure:
Duration of administration was 10-20 s
Frequency of treatment:
single administration (p.o.; i.p.)
Post exposure period:
24 hours; 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
single exposure
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
single exposure
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
single exposure
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
single exposure
No. of animals per sex per dose:
10
Control animals:
other: negative control:0.9% sodium chloride “Braun” oral gavage in the volume of 1 mL per 100 g body weight... (see attached file)
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: intraperitoneal
- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow cells (mean number of NCPE, PCE, NCE and proportion of immature erythrocytes)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Dose selection information from literature:
Insoluble tin compounds were demonstrated to have negligible toxic effects in rats when administered for 28 days in the diet. Rats were fed on diets containing 0.0, 0.03, 0.10, 0.30 or 1.00% of Tin sulfide, which did not result in adverse effects on mortality, body weight change, diet utilization, measurements of blood, urine and biochemical parameters, organ weights and gross and micropathology. The amount of 1% in the diet was calculated to correspond with approximately 670 mg/ kg body weight/day on average basis.
References:
-WHO Food Additives Series Vol:46 (2001) pp 307-60;
-de Groot A.P., Feron V. J., Til H. P. Short-term toxicity studies on some salts and oxides of tin in rats. Food and Cosmetics Toxicology, Volume 11, Issue 1, February 1973, Pages 19-30.

Dose selection information from other studies:
Information became available from an oral DRF study in preparation of the 3-month toxicity study in the rat (Exp. 32/09/C Bio Test) and from the in vitro chromosome aberration toxicity study in human peripheral lymphocytes (Exp. 31/09/C Bio Test). Based upon the information of these studies, and taking into account the literature information, dose levels were amended to 500, 1000 and 2000 mg/kg bw. The limit dose of 2000 mg/kg bw was included in this study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
-The animals were exposed to Test item and control items by single administration.
-One half of the animals from each group were sacrificed by cervical dislocation in ether anaesthesia 24 hours after the administration and the other half of animals were sacrificed 48 hours after the administration.

DETAILS OF SLIDE PREPARATION:
During the necropsy the bone marrow was extracted from femur using bovine serum into a labeled test tube. The cells were centrifuged (1000 rpm for 10 min). The supernatant was removed and the sediment was resuspended in approx. 4 drops of bovine serum. Smear preparations from this suspension were made in duplo for each animals and fixed with methanol for 5 min and then stained with Giemsa-Romanowski (1:10) for 15-20 min.
All the slides were independently coded before blind microscopic analysis.


METHOD OF ANALYSIS:
At least 2000 polychromatic (immature) erythrocytes per animal were scored for the incidence of micronuclei under the microscope. The proportion of polychromatic (immature) among total (immature + mature or polychromatic + normochromatic) erythrocytes were determined for each animals by counting a total of at least 200 erythrocytes.
Evaluation criteria:
At least 2000 polychromatic (immature) erythrocytes per animal were scored for the incidence of micronuclei under the microscope. The proportion of polychromatic (immature) among total (immature + mature or polychromatic + normochromatic) erythrocytes were determined for each animal by counting a total of at least 200 erythrocytes.
Statistics:
The analyzed data were tested for normality (Kolmogorov–Smirnov test) and homogeneity of variance
(Bartlett’s test).
The data for males and females were separately analyzed using ANOVA followed by Dunnett’s Multiple Comparison Test. The F-test in the ANOVA tests whether there are any significant differences amongst the means at the 95.0 % confidence level.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Tin sulfide does not produce micronuclei in the immature erythrocytes in the conditions of the test.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
No increase was found in the frequency of micronucleated polychromatic erythrocytes in treated animals as indication of induced chromosome damage. Tin sulfide does not induce damage to the chromosomes or the mitotic apparatus of erythroblasts.
Executive summary:

The purpose of the micronucleus test is to identify substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments or whole chromosomes.
For this study 50 male and 50 female rats (8-12 weeks old) were used, divided into 5 groups: dose group D1 (500 mg/kg), D2 (1000 mg/kg), D3 (2000 mg/kg), group C (negative control) and group CP (positive control-cyclophosphamide), each including 20 animals t 10 males and 10 females). Samples of bone marrow were taken at 24 and 48 hours after the single administration.
Each animal in treated groups received the appropriate dose of Test item suspension orally by gavage in the volume of 1 mL per 100 g body weight. The animals in the negative control group received the appropriate volume of saline solution by the same way. The animals in the positive control group were administered cyclophosphamide solution at the dose of 20 mg/kg by a single intraperitoneal application in the volume of 1 mL per 100 g body weight.
All the rats were observed for clinical signs, morbidity or mortality during acclimation, before administration and after administration in time points 0 (immediately after dosing), 0.5, 1, 2, 4 and 24 hours post-dose and then before necropsy.
All the rats were individually weighed at delivery and prior to the administration in order to calculate dose levels.
No changes in health status and condition of the animals in any of the groups were recorded during the acclimation and during the study.
No significant changes of mean body weight were observed in the animals during the study.
All the values of number of normochromatic erythrocytes with micronuclei in all dose groups (D1, D2 and D3) were within the reference range for negative control group. No statistically significant higher values of number of normochromatic erythrocytes with micronuclei in any of the dose groups (up to 11 of micronuclei) as compared to negative control group (up to 13 micronuclei) were noted. Statistically significant differences were observed in the positive control group (normochromatic erythrocytes with micronuclei -up to 38 of micronuclei) as compared to control group.

No increase was found in the frequency of micronucleated polychromatic erythrocytes in treated animals as indication of induced chromosome damage. Tin sulfide does not induce damage to the chromosomes or the mitotic apparatus of erythroblasts.