Methyl (Z,Z)-8,8-dibutyl-3,6,10-trioxo-2,7,9-trioxa-8-stannatrideca-4,11-dien-13-oate

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 from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

14 March - 11 April 1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline. Preferred study for this SIDS endpoint.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Dibutyltin dichloride

EC Number:

211-670-0

EC Name:

Dibutyltin dichloride

Cas Number:

683-18-1

IUPAC Name:

dibutyltin dichloride

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Olac Breeding Laboratories
- Age at study initiation: 4-5 weeks old
- Weight at study initiation: 17.9-25.7g
- Assigned to test groups randomly: nda
- Fasting period before study: nda
- Housing: Male and female mice were housed in single-sex groups of two or five in high density polypropylene cages with stainless steel tops (North Kent Plastics Limited), in a limited-access animal holding room. Cages housing animals of different treatment groups were distributed so that the effects of any spatially-variable components were equalised between groups as far as possible.
- Diet: Laboratory animal diet LAD 1 (Biosure, Manea, Cambridgeshire, England) was fed ad libitum throughout the study. This is an expanded autoclaved diet supplied in a discardable outer paper sack and sealed inner polythene bag. It contains no added antibiotic or other chemotherapeutic or prophylactic agent. Normal levels of common contaminants will not influence the outcome of the Study.
- Water: Drinking water was supplied to each cage via a polythene bottle and sipper-tube.
- Acclimation period: Minimum of 4 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 15%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark cycle in operation

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: The test material was found to liquify when heated to approximately 40°C in a waterbath. In this state it was found to be easily miscible with warm (40°C) corn oil at a concentration in excess of 465 mg/ml, and did not appear to come out of solution when cooled to room temperature. Consequently, dosing solutions were freshly prepared in warm corn oil on the day of dosing, each concentration being individually formulated, and mixed prior to use.
- Concentration of test material in vehicle: 465 mg/ml

All concentrations and dosages cited here refer to the DBTC sample as received; no allowance has been made for purity or activity below 100%.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was found to liquify when heated to approximately 40°C in a waterbath. In this state it was found to be easily miscible with warm (40°C) corn oil at a concentration in excess of 465 mg/ml, and did not appear to come out of solution when cooled to room temperature. Consequently, dosing solutions were freshly prepared in warm corn oil on the day of dosing, each concentration being individually formulated, and mixed prior to use.

Duration of treatment / exposure:

Male and female mice were given a single dose of DBTC at 2, 10 or 50 mg/kg. In all cases DBTC was dosed orally, mixed with corn oil. Five males and five females from each group were scheduled for termination 24 hours after treatment; further lots of five males and five females, given DBTC at 50 mg/kg or the vehicle control, were scheduled for termination 48 and 72 hours after treatment.

Frequency of treatment:

Male and female mice were given a single dose of DBTC at 2, 10 or 50 mg/kg.

Post exposure period:

Animals were inspected daily throughout the acclimatisation period and the dosing period for signs of ill-health or reaction to treatment. Any deviation from normal was recorded. All animals were weighed on the day of treatment and again immediately before termination, and bodyweights were recorded.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
2 mg/kg
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
10 mg/kg
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
50 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

15 males and 15 females (30 in total) were used in the corn oil controls group and the group which ingested 50 mg/kg of the test material.
5 males and 5 females (10 in total) were used for all other concentrations and the positive control.

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control substance: Chlorambucil
- Justification for choice of positive control(s): nda
- Route of administration: oral
- Doses / concentrations: 30 mg/kg in aqueous 10% ethanol.

Examinations

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The preliminary toxicity test was conducted using DBTC dosages of 62.5, 125.0, 250.0 and 500.0 mg/kg.
All animals dosed with DBTC at 125, 250 and 500 mg/kg showed adverse reactions to treatment including severe rales, piloerection, immobility, hunched posture and uneven respiration: all were killed in extremis 4 hours (500 mg/kg) or 23 hours (125 and 250 mg/kg) following dosing, and all were seen to have lost weight since dosing. All animals dosed at 62.5 mg/kg showed piloerection on the day following dosing, and both males were hunched and lethargic from day 3 until termination: all these animals lost weight over the 72 hour period.
Slides were prepared and stained for all animals. Examination of slide preparations showed evidence of bone marrow toxicity (depression in bone marrow proliferation) in individual animals dosed at 62.5, 125.0 or 250.0 mg/kg.
After consideration of these data, the highest DBTC dosage selected for the main micronucleus test was 50 mg/kg.


TREATMENT AND SAMPLING TIMES:
Male and female mice were given a single dose of DBTC at 2, 10 or 50 mg/kg. In all cases DBTC was dosed orally, mixed with corn oil. Five males and five females from each group were scheduled for termination 24 hours after treatment; further lots of five males and five females, given DBTC at 50 mg/kg or the vehicle control, were scheduled for termination 48 and 72 hours after treatment.


DETAILS OF SLIDE PREPARATION:
Animals were killed by cervical dislocation following carbon dioxide inhalation. Femurs from each animal were rapidly dissected out and cleaned of adherent tissue. The epiphyses were cut off to obtain access to the marrow canal. Marrow cells were flushed out with 2.5 ml foetal calf serum using a syringe and needle. The recovered cells were centrifuged at 1000 rpm for five minutes. The bulk of the supernatant fluid was discarded and the cell pellet resuspended in the remaining fluid. Single drops of the cell suspension were transferred to clean, dry slides, two or three smears (for the preliminary toxicity test or main micronucleus test respectively) prepared, and the slides left to air-dry. Following fixation in methanol for ten minutes, they were stained manually, using the Schmid (May-Grunwald and Giemsa) staining technique.

When air-dried, permanent mounts were made using DPX mountant, after clearing for five minutes in xylene.


METHOD OF ANALYSIS:
At least one slide from each animal was randomly coded by a person not subsequently involved in the scoring of the study. Care was taken to ensure that no unique slide identifications remained visible in order to eliminate bias.
Slides were examined under the light microscope, and regions judged to be of adequate technical quality to permit scoring were selected under low magnification. At high magnification (x 1000, oil immersion) a total of at least 2000 erythrocytes per animal were examined. Each erythrocyte scored was classed as polychromatic or mature: polychromatic cells stain blue/pink and the older cells stain red/pink. At least 1000 cells of each type were scored from each animal where possible, but where there was an appreciable deviation from unity in the ratio of polychromatic to mature erythrocytes, scoring continued until a minimum of 2000 of the predominant cell type were counted. In addition each erythrocyte scored was examined for the presence or absence of micronuclei. When examination had been completed, the data were decoded.

The frequencies of micronucleated cells per 1000 erythrocytes were then calculated. The ratio of polychromatic to mature cells was also determined; a decrease in this may indicate inhibition of cell division following treatment, and the incidence of micronuclei in the mature cell population 24 hours after treatment reflects the pretreatment situation, since most of these cells were produced before treatment. The frequency of micronuclei in polychromatic cells provides an index of induced genetic damage.

Evaluation criteria:

Slide evaluation was restricted to determination of numbers of polychromatic and mature erythrocytes and calculation of the ratio of polychromatic to mature cells for each animal, and for each group.

Statistics:

The frequencies of micronucleated cells per 1000 polychromatic erythrocytes scored were subjected to statistical analysis by the Mann-Whitney procedure (Mann and Whitney, 1942). A computer-based version of this test was employed and significance was determined by reference to tabulated values of R1.
Data from males and females within each group were compared using a two-tailed test. Where there was no significant difference within the group, the sexes were pooled for further analysis. For each sampling time (24, 48 or 72 hours), each treated group was compared with concurrent vehicle controls using a one-tailed test.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The preliminary toxicity test was conducted using DBTC dosages of 62.5, 125.0, 250.0 and 500.0 mg/kg.
All animals dosed with DBTC at 125, 250 and 500 mg/kg showed adverse reactions to treatment including severe rales, piloerection, immobility, hunched posture and uneven respiration: all were killed in extremis 4 hours (500 mg/kg) or 23 hours (125 and 250 mg/kg) following dosing, and all were seen to have lost weight since dosing. All animals dosed at 62.5 mg/kg showed piloerection on the day following dosing, and both males were hunched and lethargic from day 3 until termination: all these animals lost weight over the 72 hour period.
Slides were prepared and stained for all animals. Examination of slide preparations showed evidence of bone marrow toxicity (depression in bone marrow proliferation) in individual animals dosed at 62.5, 125.0 or 250.0 mg/kg.


RESULTS OF DEFINITIVE STUDY
At a dosage of 2 mg/kg, no animal showed reactions to treatment. At 10 mg/kg, 3 males showed hunched posture and piloerection on the day of dosing only: no signs were observed in females. No marked incidences of weight loss were apparent in animals of either group. At 50 mg/kg, one male was killed in extremis approximately 2 hours after dosing (as a result of inactivity, unstable gait, slow respiration and piloerection). All but one of the remaining animals showed reactions to treatment including hunched posture, piloerection, inactivity, rales, closing of one or both eyes, and yellow staining of the coat. In addition, one female was found dead at termination, although it was seen to be alive 2 hours previously. At the 24 hour termination time, 5 animals had lost weight and one had failed to gain weight. At the 48 hour termination time all animals were seen to have lost weight, and all but two animals had lost weight at the 72 hour termination time. All weight losses recorded at 48 and 72 hours were marked. Of the ten mice given chlorambucil, the positive control agent, seven lost weight during the 24 hour period before termination.

Any other information on results incl. tables

Among mice killed 24 hours after treatment, the mean incidence of micronucleated polychromatic erythrocytes (per 1000 polychromatic cells scored) was 1.6 for the vehicle control group, with a range of 0.0-3.9. Corresponding values for animals given DBTC at 2, 10 or 50 mg/kg were closely similar: 1.8, 1.7 or 1.0 with ranges of 0.0-4.2, 0.0-4.6 and 0.0-4.0 respectively.

Thus, mean values for DBTC treated groups were closely similar to mean control group values at the 24 hour termination time, and statistical analysis confirmed that there was no significant difference between the vehicle control group and any DBTC treated group (p>0.05).

Among mice killed after 48 or 72 hours, the mean incidences of micronucleated polychromatic erythrocytes in vehicle controls were 0.6 (range 0.0-2.0) and 1.4 (range 0.0-2.9) respectively. Corresponding values for animals given DBTC at 50 mg/kg were 3.5 (range 0.0-17.1) and 5.3 (range 0.0-17.5) respectively.

Thus marked increases in the mean frequency of micronucleated polychromatic cells were apparent in animals dosed with DBTC at 50 mg/kg at both termination times. It should be noted that these increases were principally due to data from female mice at both 48 and 72 hours, and only one male at the 72 hour termination showed a marked increase in micronucleated polychromatic cells (although this animal also showed marked bone marrow toxicity). However, statistical analysis showed no significant differences between sexes in the frequency of micronucleated polychromatic cells at either termination time, and data were pooled for comparison to concurrent vehicle control data, which showed the observed increases at both termination times to be statistically significant (0.05>p>0.01 in both cases).

Chlorambucil treatment produced a range of micronucleated cells per 1000 polychromatic erythrocytes from 34.0 to 75.8 with a mean of 49.7. Statistical analysis showed that animals treated with chlorambucil had significantly more micronucleated polychromatic cells than vehicle controls (p<0.01). This increase in chromosomal damage after exposure to a known mutagen demonstrates the sensitivity of the test system.

The recorded incidence of micronuclei per 1000 mature erythrocytes varied between 0.0 and 2.9 throughout all groups.

The ratio of polychromatic to mature erythrocytes was 1.0 in the vehicle control group at 24 hours. Ratios for groups given DBTC at 2, 10 or 50 mg/kg and terminated 24 hours later were 1.1, 1.1 or 1.0 respectively. Forty-eight hours after treatment, the ratio of the vehicle control group and the group given DBTC at 50 mg/kg was 0.8. Seventy-two hours after treatment, the ratio of polychromatic to mature erythrocytes in the vehicle control group was 0.7, and in DBTC treated animals it was 0.5.

Thus no evidence of bone marrow toxicity was observed in animals treated with DBTC and killed 24 or 48 hours later; at 72 hours, although the vehicle control group ratio was slightly reduced (0.7), a marked reduction was observed in animals treated with DBTC at 50 mg/kg (0.5). In animals treated with chlorambucil, the ratio between polychromatic and mature erythrocytes was 0.8.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): positive
It is concluded that, under the conditions of test, there was evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of mice treated orally with DBTC at 50 mg/kg and sacrificed 48 or 72 hours later.

Executive summary:

In an assessment of clastogenic action on bone marrow erythrocytes in the micronucleus test (Life Science Research Limited project number: 91/0357) the test material showed evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of mice treated orally with DBTC at 50 mg/kg and sacrificed 48 or 72 hours later.

The procedures and experimental design employed complied with the recommendations of the relevant toxicity testing guideline of the OECD (Guideline 474, 1983), the US EPA Guidelines (1985) and EEC Annex V Method B.12.

A biologically and statistically significant increase in the incidence of micronucleated polychromatic cells was observed in the bone marrow of mice treated with DBTC at 50 mg/kg and killed 48 and 72 hours later (0.01<p<0.05): this effect was seen more clearly in females than in males. No such effect was apparent for any group treated with DBTC and killed 24 hours later (p>0.05). Statistically significant increases over controls were also seen in positive control group animals given chlorambucil at 30 mg/kg (p<0.01).