Diisooctyl 2,2'-[(dioctylstannylene)bis(thio)]diacetate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

no

Type of assay:

other: mammalian erythrocyte micronucleus test (migrated information)

Test material

Test material information

Constituent 1

Reference substance name:

Diisooctyl 2,2'-[(dioctylstannylene)bis(thio)]diacetate

EC Number:

247-666-0

EC Name:

Diisooctyl 2,2'-[(dioctylstannylene)bis(thio)]diacetate

Cas Number:

26401-97-8

Molecular formula:

C36-H72-O4-S2-Sn

IUPAC Name:

6-methylheptyl 14-methyl-4,4-dioctyl-7-oxo-8-oxa-3,5-dithia-4-stannapentadecan-1-oate

Constituent 2

Reference substance name:

Mono-n-octyltin-tri-thioglycol-acid isooctyl ester

IUPAC Name:

Mono-n-octyltin-tri-thioglycol-acid isooctyl ester

Test material form:

liquid

Details on test material:

- Appearance: colourless liquid

Test animals

Species:

mouse

Strain:

other: CFLP

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 19 to 23 g
- Assigned to test groups randomly: yes
- Fasting period before study: animals were starved overnight before dosing
- Housing: each group of 5 mice were kept in a plastic disposable cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2 °C
- Humidity: 50 ± 5 %
- Air changes (per hr): 20
- Photoperiod: The room was illuminated by artificial light for 12 hours per day

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Dosing volume: 0.1 mL/10 g bw per dose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Preparation: The formulations were prepared using a Silverson high speed mixer.

Duration of treatment / exposure:

The total doses were given as two equal administrations separated by an interval of 24 hours.

Frequency of treatment:

Two equal administrations separated by an interval of 24 hours.

Post exposure period:

6 hours following the second dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 250 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

4 500 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

9 000 mg/kg bw (total dose)

No. of animals per sex per dose:

Phase I (preliminary toxicity study): 2 animals per sex per dose
Phase II (micronucleus study): 5 animals per sex per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

- Mitomycin C
- Doses / concentrations: Prepared at 0.7 mg/mL for two doses of 7 mg/kg, in 0.9 % saline
- Route of administration: Intra-peritoneally

Examinations

Tissues and cell types examined:

- The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic and 2000 normochromatic erythrocytes per animal.
- In addition, the ratio of normochromatic to polychromatic erythrocytes was determined for each animal.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
- Doses for the micronucleus assay were selected from the results of the preliminary toxicity study.

DETAILS OF SLIDE PREPARATION:
- Following the last dose, the animals were observed for a further six hours before killing and all mortalities and signs of malreaction during the experiment were recorded. The animals were killed by cervical dislocation and both femurs dissected out from each animal. The femurs were cleared of tissue and one epiphysis removed from each bone.
- A direct bone marrow smear was made onto a slide containing a drop of calf serum. The slide was cleaned by immersion in methanol for 24 hours and air-dried immediately before use. One smear was made from each femur. The prepared smears were air-dried and fixed in methanol overnight.
- After fixation, the smears were air-dried and placed in buffered distilled water (pH 6.8) for 10 minutes prior to staining in Giemsa (dilution factor 1 part Giemsa: 9 parts buffered distilled water pH 6.8) for 10 minutes. After rinsing in buffered distilled water (pH 6.8), the slides were air-dried. Prior to mounting in DPX, the slides were defatted by immersion in xylene for 10 minutes.

METHOD OF ANALYSIS:
- The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic and 2000 normochromatic erythrocytes per animal. In addition, the ratio of normochromatic to polychromatic erythrocytes was determined for each animal.

Statistics:

STATISTICAL ANALYSIS OF THE RATIOS OF NORMOCHROMATIC TO POLYCHROMATIC ERYTHROCYTES
- Due to heterogeneity of variance (Barlett's test; P < 0.001) non-parametric methods based on Kruskal-Wallis mean ranks were used to analyse the ratios of normochromatic to polychromatic erythrocytes. These methods have been found to be more robust against inequality of variance than classical analysis of variance methods.
- The positive control group dosed with Mitomycin C and the groups dosed with the test material were compared with the vehicle control using the non-parametric equivalent of the method of L.S.D.'s (least significant differences).
- After administration of the test material at a total dosage of 2250 mg/kg bodyweight, the ratio of normochromatic to polychromatic erythrocytes was not significantly different from the concurrent control value. At total dosages of 4500 and 9000 mg/kg body weight the ratios were significantly different from the concurrent control value, P < 0.01 and P < 0.001 respectively
- The positive control group dosed with Mitomycin C, gave a ratio which was significantly different from the concurrent control value, P < 0.001.

STATISTICAL ANALYSIS OF THE MICRONUCLEATED NORMOCHROMATIC AND POLYCHROMATIC ERYTHROCYTE COUNTS
- The non-parametric methods of statistical analysis detailed above were used to analyse the micronucleated normochromatic and polychromatic erythrocyte counts.
- After administration of the test material at all dosages, the micronucleated normochromatic and polychromatic erythrocyte counts were not significantly different from the respective concurrent control values.
- The positive control group dosed with Mitomycin C gave micronucleated cell counts which were significantly different from the respective concurrent control values, P < 0.001

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Bone marrow depression

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000, 4000, 8000, 9000, 10000, 11000, 12000, 14000 and 20000 mg/kg bw
- Clinical signs of toxicity in test animals: No toxic reactions were observed at 2000, 4000, 8000 and 9000 mg/kg bw. At 10000 and 11000 mg/kg bw, a toxic reaction consisting of hypopnoea was observed. At total dosages of 12000, 14000 and 20000 mg/kg bw, a toxic reaction consisting of hypopnoea and a period of lethargy was observed.
- From the above results, a top dosage of 9000 mg/kg bodyweight was chosen for the micronucleus test which, it was indicated, would cause one or two deaths.

RESULTS OF DEFINITIVE STUDY

SIGNS AND MORTALITIES
- After administration of 1 % methylcellulose, the vehicle control, no toxic reactions or mortalities were observed.
- After administration of the test material at all dosages, no toxic reactions were observed. At a total dosage of 9000 mg/kg bodyweight, there were three mortalities. Two animals died overnight after the administration of the first dose, between 8 and 24 hours. Advanced visceral autolysis prevented any post-mortem examination. The third died after administration of the second dose between 24 and 30 hours. Macroscopic examination at post-mortem failed to reveal any abnormalities.
- After administration of Mitomycin C, the positive control compound, no toxic reactions or mortalities were observed.

MICRONUCLEUS COUNTS
- The group mean counts and ranges obtained with the test material are shown in Table 1.
- In this experiment the negative control group gave a mean count of 2.8 micronucleated polychromatic cells which was just outside the range for previous unrelated experiments. However, the individual counts were within the laboratory standard range of individual counts for negative controls obtained in previous unrelated experiments.
- After administration of the test material at all dosages, the group mean polychromatic cell counts were comparable with the concurrent control value and within the laboratory standard range for negative controls obtained in previous unrelated experiments. In addition, the group mean micronucleated normochromatic cell counts of the treatment groups were comparable with the concurrent control value.
- The positive control, Mitomycin C, administered intraperitoneally gave a mean count of 89.5 micronucleated cells per 2000 polychromatic erythrocytes. This group also gave a mean count of 11.4 micronucleated cells per 2000 normochromatic erythrocytes.

Any other information on results incl. tables

Table 1: Group mean number of micronucleated cells per 2000 normochromatic erythrocytes and 2000 polychromatic erythrocytes per animal and the group mean ratio of normochromatic to polychromatic erythrocytes

Treatment	Total dosage over 24 h (mg/kg)	Number of micronucleated cells per 2000 erythrocytes per animal				Ratio of normochromatic to polychromatic erythrocytes
		Normochromatic		Polychromatic
		Mean	Range	Mean	Range	Mean	Range
Vehicle	-	2.5	0 to 6	2.8	1 to 6	1.09	0.93 to 1.22
Test material	2250	2.3	0 to 6	2.5	0 to 5	1.38	1.19 to 1.69
	4500	2.4	0 to 5	2.4	0 to 6	1.72**	1.47 to 2.09
	9000	2.3	0 to 6	2.6	1 to 5	3.93***	2.56 to 5.39
Positive control	14	11.4***	8 to 15	89.5***	74 to 115	10.46***	7.75 to 15.38

** P < 0.01

*** P < 0.001

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, it was concluded that the test material failed to show any evidence of mutagenic potential, when administered orally. However, at the higher dose levels evidence of toxicity, as shown by bone marrow depression, was observed.

Executive summary:

A study was performed to investigate the genetic toxicity in vivo of the test material. The study was conducted in a style similar to OECD 474.

In this assessment of the effect of the test material on the incidence of micronucleated polychromatic erythrocytes in mice, total dosages of 2250, 4500 and 9000 mg/kg bodyweight were administered by oral gavage, in two equal dosages, separated by an interval of 24 hours.

A negative control group was dosed in an identical manner with the vehicle, 1 % methylcellulose. A positive control group was dosed by intraperitoneal injection with Mitomycin C, at a total dosage of 14 mg/kg bodyweight. The mice were sacrificed six hours after the second dose and bone marrow smears examined for the presence of micronuclei in 2000 polychromatic and 2000 normochromatic erythrocytes per mouse. The ratio of normochromatic to polychromatic cells was also examined in each mouse.

A preliminary toxicity study indicated that a top dosage of 9000 mg/kg bodyweight would cause one or two deaths and led to the selection of the dose concentrations for the main test.

At all dosages of the test material both of the group mean micronucleated cell counts were comparable with the concurrent control values.

After administration of the test material at a total dosage of 2250 mg/kg bodyweight, the ratio of normochromatic to polychromatic cells was comparable with the concurrent control value. At total dosages of 4500 and 9000 mg/kg bodyweight, the ratios were higher than the concurrent control value. Statistical analysis, using Kruskal-Wallis methods, showed these increases to be significantly different. The positive control compound, Mitomycin C, produced the expected large increase in the group mean polychromatic micronucleated cell count and in the normochromatic to polychromatic cell ratio. This group also produced a small increase in the group mean micronucleated normochromatic cell count.

Under the conditions of this study, it was concluded that the test material failed to show any evidence of mutagenic potential, when administered orally. However, at the higher dose levels evidence of toxicity, as shown by bone marrow depression, was observed.