2-ethylhexyl 10-ethyl-4,4-dimethyl-7-oxo-8-oxa-3,5-dithia-4-stannatetradecanoate

Genetic toxicity: in vivo

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: Preferred study for this SIDS endpoint. Performed in compliance with USEPA, TSCA, and OECD Good Laboratory Practices (GLP).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: ASTM Standard No. E1263-88

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyltin dichloride

EC Number:

212-039-2

EC Name:

Dimethyltin dichloride

Cas Number:

753-73-1

Molecular formula:

C2H6Cl2Sn

IUPAC Name:

dimethyltin dichloride

Constituent 2

Reference substance name:

Stannane, dichlorodimethyl-

IUPAC Name:

Stannane, dichlorodimethyl-

Details on test material:

- Name of test material (as cited in study report): Mixes of methyltin compounds
- Molecular formula (if other than submission substance): C2H6Cl2Sn
- Molecular weight (if other than submission substance): 219.67 g/mol
- Smiles notation (if other than submission substance): C[Sn](C)(CL)CL
- Physical state: solid
- Analytical purity: >99.9%
- Composition of test material, percentage of components:
- Lot/batch No.: 1561-122
- Stability under test conditions: stable
- Storage condition of test material: stored in a secondary container at room temperature

Test animals

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: approx. 5 weeks
- Weight at study initiation: 15.6 to 24 grams
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: no more than 10 to a cage during quarantine; 3 to a cage for the range-finding; 5 to a cage during the test
- Diet (e.g. ad libitum): Purina Certified Rodent Chow #5002; ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25.6 deg F
- Humidity (%): 13-67%
- Photoperiod (hrs dark / hrs light): 12 hours light/ 12 hours dark


IN-LIFE DATES: From: 1990-10-29 To: 1990-12-08

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: test substance solubility
- Concentration of test material in vehicle: 10 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed well in deionized water immediately before dosing and administered by gavage. The volume of test solution administered was 10 mL/kg bw.

Duration of treatment / exposure:

0, 24, 48, and 72 hours

Frequency of treatment:

single dose

Post exposure period:

animals were sacrificed at approximately 24, 48 and 72 hours post-dose.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 200, and 400 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Positive control(s):

urethane
- Justification for choice of positive control(s): to ensure proper conduct of the assay procedures
- Route of administration: gavage; to male mice only
- Doses / concentrations: 300 mk/kg bw

Examinations

Tissues and cell types examined:

Bone Marrow analysis: The right femur from each mouse was removed and  flushed with 0.2 mL of fetal bovine serum (FBS) into 0.5 mL of FBS in a  2-mL conical polycarbonate tube.  Cells were concentrated by  centrifugation, then resuspended in an equal volume of supernate.  The  sample was transferred to 3 slides per animal, spread, air-dried, fixed  in absolute methanol for 5 minutes, and stored until staining.  Two of  the three slides were coded.  Immediately before scoring, the remaining  slide was stained with acridine orange.

Peripheral blood analysis: Peripheral blood was analyzed for the PCE/RBC  ratio in the range finding test only.  Blood samples were obtained from  the ventral tail vessel and transferred to 3 slides/mouse, spread,  air-dried, fixed in absolute methanol for 5 minutes, and stored until  staining.  Two of the 3 slides were coded.  Immediately before scoring,  the remaining slide from each animal was stained with acridine orange.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Dose levels selected were based on a preliminary range finding test.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Mice were dosed once and sacrificed approximately 24, 48 and 72 hours after dose administration.


DETAILS OF SLIDE PREPARATION: Cells were concentrated by  centrifugation, then resuspended in an equal volume of supernate.  The  sample was transferred to 3 slides per animal, spread, air-dried, fixed  in absolute methanol for 5 minutes, and stored until staining.  Two of  the three slides were coded.  Immediately before scoring, the remaining  slide was stained with acridine orange.

Blood samples were obtained from  the ventral tail vessel and transferred to 3 slides/mouse, spread,  air-dried, fixed in absolute methanol for 5 minutes, and stored until  staining.  Two of the 3 slides were coded.  Immediately before scoring,  the remaining slide from each animal was stained with acridine orange.

METHOD OF ANALYSIS: Peripheral blood and bone marrow smears were evaluated using  epifluorescence microscopy.  Two parameters were determined in the bone  marrow smears: (1) number of micronucleated RNA-positive erythrocytes per  animal, and (2) number of RNA-positive erythrocytes in 200 erythrocytes  per animal.  Data were analyzed both separately (by sex) and together,  unless a statistically significant sex difference was observed in the  vehicle control groups.  The ratio of micronucleated RNA-containing  erythrocytes (i.e., micronucleated PCE) to RNA-positive erythrocytes and  the percentage of RNA-positive erythrocytes among total erythrocytes were  calculated for each animal.  The determination of frequencies of micronucleated cells was based on  scoring 1000 cells per animal.  The data were not expected to be normally  distributed.  


OTHER:

Evaluation criteria:

The data from this assay were considered acceptable if: the frequency of micronucleated cells in the vehicle control group was within the normal historical range; administration of the positive control substance resulted in a statistically significant elevation of micronucleated cells, and; there were at least three surviving animals of each sex with a percentage of RNA-positive erythrocytes greater than or equal to 15% of the control value.

Statistics:

Data were analyzed both separately by sex and together unless a statistically significant sex difference was observed in the vehicle control groups. The ratio of micronucleated RNA-containing erythrocytes to RNA-positive erythrocytes and the percentage of RNA-positive erythrocytes among total erythrocytes were calculated for each animal. The statistical significance of differences in the percentage of RNA-positive erythrocytes among groups was evaluated using the Kruskall-Wallace analysis of variance on ranks. If an overall difference between the control groups and any individual dose group was observed, the significance of the difference was determined by using a distribution-free multiple comparison test.

In experiments where the determination of frequencies of micronucleated cells is based on scoring 1000 cells per animal, data are not expected to be distributed normally. Such data were analyzed using the Cochran-Armitage test for trends in binomial proportions and the normal test for equality of proportions.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 0, 150, 300, 600, 1200 and 2500 mg/kg bw
- Solubility: mixed well with deionized water
- Clinical signs of toxicity in test animals: humped backs, rough fur, labored breathing, diarrhea, lacrimation and moribundity. All animals died in the three top dose groups.
- Evidence of cytotoxicity in tissue analyzed: yes
- Rationale for exposure: to maximize exposure of the animal to the test substance
- Harvest times: animals were sacrificed approximately 72 hours after single dose administration.



RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay):
% PCE/RBC VALUES (0-72 h):
Control (0 mg/kg bw):
Males, 54.3-54.2 F
emales, 53.2-54.5 

100 mg/kg bw:
Males, 51.3-51.8
Females, 50.6-51.4

200 mg/kg bw:
Males, 44.0-46
Females, 43-43.4

400 mg/kg bw:
Males, 42.9-42.4
Females, 38.8-37.5

Urethane (300 mg/kg bw):
Males only, 38.9-50.1

PCE suppression was observed in females in the 400 mg/kg bw dose group. 

% PCE with micronuclei (0-72 h):

Control (0 mg/kg bw):
Males, 0.20-0.20
Females, 0.18-0.18

100 mg/kg bw:
Males, 0.16-0.18
Females, 0.20-0.22

200 mg/kg bw:
Males, 0.20-0.18
Females, 0.18-0.25

400 mg/kg bw:
Males, 0.10-0.24
Females, 0.10-0.20

Urethane (300 mg/kg bw):
Males, 2.43-0.65

- Appropriateness of dose levels and route: The PCE/RBC ratio in the bone marrow assay at 300 mg/kg bw was more than 50% of the control PCE ratio. Because all animals died at the nxt three higher dose levels, 400 mg/kg bw was chosen as the high dose for the definitive. The route was chosen to maximize exposure of the animals to the test substance.

Any other information on results incl. tables

MORTALITY (number of deaths/number of animals tested)
0 mg/kg bw: Males, 0/15; Females, 0/15
100 mg/kg bw: Males, 0/15; Females 0/15
200 mg/kg bw: Males, 3/15; Females, 3/15
400 mg/kg bw: Males, 3/15; Females, 8/15

Deaths were preceded by clinical signs of systemic toxicity, including  humped backs, rough fur, diarrhea, and labored breathing.  Surviving mice  at 200 and 400 mg/kg bw dose levels exhibited similar clinical signs.   Three males at 100 mg/kg bw had persistent tremors and 2 of the 3 had  rough fur.  All female mice at 100 mg/kg bw appeared normal throughout  the assay.  At 200 mg/kg bw, 1 male had rough fur; 1 male had rough fur,  humped back, and diarrhea; and 2 males had rough fur and humped backs.   Four females at 200 mg/kg bw had rough fur, humped backs, and persistent  tremors beginning on Day 4.  At 400 mg/kg bw, 4 males and rough fur; 2  males had rough fur and humped backs; 2 males had rough fur, humped  backs, and diarrhea; and 1 male had rough fur and tremors.  Three females  at 400 mg/kg bw had rough fur, and 3 other females had rough fur and  humped backs.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test substance did not induce micronuclei in polychromatic  erythrocytes from Swiss-Webster mice under the conditions of the assay.

Executive summary:

The ability of the test substance to induce micronuclei (MN) in bone marrow erythrocytes follwoing oral administration in mice was assessed. A range-finding assay was performed to determine the doses to be used in the definitive study. On the basis of the incidence of mortality, the doses selected for the definitive assay were 100, 200 and 400 mg/kg bw. The positive control selected for the definitive assay, administered to male mice only, was urethane (300 mg/kg).

Mice treated with deionized water at 24, 48 or 72 hours before sacrifice in the definitive assay yielded a MN frequency of 0.20, 0.12 and 0.20%, respectively, for males and 0.18% at all three time points for females. Doses of 0, 100, 200 and 400 mg/kg bw of the test substance yielded a percentage of PCE with MN of 0.24% or less in males and 0.25% or less in females. In contrast, the positive control administered 24, 48 or 72 hours before sacrifice yielded 2.43%, 1.29% and 0.65% PCE with MN, respectively, in males.

On the basis of these data, it was concluded that the test substance does not induce an increase in micronuclei in bone marrow erythrocytes.