Trichlorooctylstannane

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29th November to 2nd December 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study did not appear to have been conducted in accordance with Good Laboratory Practices (GLP); however, quality assurance was equivalent.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

Cell cycle arrest not reported

Principles of method if other than guideline:

15 male and 15 female NMRI mice per group were given once by gavage 0.5, 1.0 or 2.0 g ZK 26.384/kg body weight. Control animals (15 males and 15 females) were given the vehicle at 10 ml/kg in the same manner. Triaziquone (0.15 mg/kg; single i.p. treatment) served as positive control (5 males, 5 females).
5 males and 5 females from the negative control and the ZK 26.384 groups were killed at intervals of 24, 48 and 72 hours after treatment. The positive control animals were killed 24 hours after treatment. Femur bone marrow smears were prepared and stained using May-Gruenwald and Giemsa solutions.
The coded slides were examined for the presence of micronuclei in 2000 polychromatic and 1000 normochromatic erythrocytes and for the ratio of polychromatic to normochromatic erythrocytes.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

ZK 26.384

IUPAC Name:

ZK 26.384

Constituent 2

Reference substance name:

Mono-n-octyltin trichloride

IUPAC Name:

Mono-n-octyltin trichloride

Details on test material:

- Name of test material (as cited in study report): ZK 26.384
- Lot/batch No.: W88/121
- Expiration date of the lot/batch: According to the label the batch is stable until Nov. 1989
- Stability under test conditions: The compound is not expected to be stable in aqueous solutions for 8h, according to "Auftrag zur Durchfiihrung einer Vertraglichkeitsprufung", dated Nov. 21, 1988.
- Storage condition of test material: Can be stored in closed containers at room temperature in a dry place. The substance is sensitive to hydrolysis.

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SAVO-Ivanovas, Kisslegg, FRG.
- Age at study initiation: 10 to 11 weeks old at treatment
- Weight at study initiation: Males: 35-39 g; Females: 26-31 g
- Assigned to test groups randomly: Yes, under following basis: by lot
- Housing: They were housed individually in Macrolon type II cages containing wood chip bedding.
- Diet (e.g. ad libitum): A commercial rodent diet (Altromin R; Altromin, Lage, FRG) was supplied ad libitum.
- Water (e.g. ad libitum): Tap water was supplied ad libitum.
- Acclimation period: The animals were acclimatized in our animal house for at least 10 days before use on test.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21 °C
- Humidity (%): 54-57%
- Air changes (per hr): Kept in an air-conditioned room
- Photoperiod (hrs dark / hrs light): The room was illuminated by artificial light for 12 hours per day (c. 6.00-18.00).

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Arachis oil
The compound is not expected to be stable in aqueous solutions for 8h, according to "Auftrag zur Durchfiihrung einer Vertraglichkeitsprufung",
dated Nov. 21, 1988. Stability in arachis oil is unknown. Batch no. 176-1 (50 mg/ml) contained 109.8% of declaration (Analysis KIAN No. 2266)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The solutions in arachis oil contained 50, 100 and 200 mg ZK 26.384/ml and were prepared on the morning of treatment.
The compound is not expected to be stable in aqueous solutions for 8h, according to "Auftrag zur Durchfiihrung einer Vertraglichkeitsprufung",
dated Nov. 21, 1988. Stability in arachis oil is unknown. Batch no. 176-1 (50 mg/ml) contained 109.8% of declaration (Analysis KIAN No. 2266)

Duration of treatment / exposure:

Three doses of ZK 26.384, namely 0.5, 1.0 and 2.0 g/kg were given by gavage once in the morning until noon to 15 randomly selected (by lot) male and female mice (application volume: 10 ml/kg). Control animals (15 males and 15 females) were given the vehicle at 10 ml/kg in the same manner.

Frequency of treatment:

Single oral dose

Post exposure period:

nda

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.5 g/kg
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1.0 g/kg
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
2.0 g/kg
Basis:
actual ingested

No. of animals per sex per dose:

Three doses of ZK 26.384, namely 0.5, 1.0 and 2.0 g/kg were given by gavage once in the morning until noon to 15 randomly selected (by lot) male and female mice.

Control animals:

yes, concurrent vehicle

Positive control(s):

Triaziquone (0.15 mg/kg, 10 ml/kg, single i.p. treatment) served as positive control (5 males, 5 females).
- Positive control vehicle(s)/solvent(s) used: physiological saline
- Concentration of test material in vehicle: 0.015 mg/ml
- Lot/batch no. (if required): NAW 34B

Examinations

Tissues and cell types examined:

The slides were examined for the incidence of micronucleated cells per 2000 polychromatic (PCE) and 1000 normochromatic (NCE) erythrocytes per animal. The ratio of polychromatic to normochromatic erythrocytes was calculated on the basis of 1000 NCE scored.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
It was assumed from a preceeding acute toxicity study in male and female mice that 2.0 g/kg would be a dose level at which toxic effects might be noted (at 3 g/kg three out of six animals died).


DETAILS OF SLIDE PREPARATION:
5 males and 5 females from the negative control and the ZK 26.384 groups were killed by cervical dislocation at intervals of 24, 48 and 72 hours after treatment (the positive control animals were killed 24 h after treatment) and both femurs dissected out from each animal. The bone marrow was flushed/aspirated into fetal calf serum. The resulting cell suspensions were centrifuged and smears were prepared from drops of the cell pellets which had been resuspended in a few drops of serum. The slides were air-dried and stained using May-Gruenwald and Giemsa solutions.


METHOD OF ANALYSIS:
The slides were coded and analyzed "blind" in random order. The stained smears were examined using oil immersion high power magnification in regions where cells were well spread and stained.
The slides were examined for the incidence of micronucleated cells per 2000 polychromatic (PCE) and 1000 normochromatic (NCE) erythrocytes per animal. The ratio of polychromatic to normochromatic erythrocytes was calculated on the basis of 1000 NCE scored.

Evaluation criteria:

The purpose of this study was to investigate whether ZK 26.384 can induce chromosome breakage or malfunction of the spindle apparatus leading to aneuploidy in vivo measured by the increase in micronucleated erythrocytes in mice.

If scoring is restricted to polychromatic erythrocytes, all the damage detected will have been caused during the final cell cycle of the nucleated precursor cells. Thus by examining polychromatic cells at each kill, the effect of the compound over the previous c. 24 hours can be monitored.

Statistics:

The statistical analysis was conducted for each of the following variables:
p1: proportion of micronucleated PCE
p2: proportion of micronucleated NCE
p3: ratio of PCE/NCE

The variables p1 and p2 were arc sin pi transformed. The analyses were conducted separately for the sample times. Regarding the first sample time a two-factorial analysis of variance (ANOVA) with the factors "sex" and "treatment" was performed to assess the difference between positive and negative controls with pooled values for both sexes; thereafter the positive control group was excluded from further analysis. Thereafter in another two-factorial ANOVA (factors "sex", "treatment") for each sample time it was investigated if there was a treatment effect. In case of significant interactions, comparisons between the control and each of the treatment groups were conducted separately for the sexes. If there were no significant interactions but a global treatment effect, comparisons were performed with values pooled for both sexes. The pairwise comparisons were performed with one-sided t-tests (increase of p1 and p2, decrease of p3), using the error estimate of the ANOVA table. The test levels were always a = 0.05 (least significant differences test-LSD).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay):
From the results obtained, it was concluded that ZK 26.384 failed to produce any increase in the number of micronucleated polychromatic or normochromatic erythrocytes in male and female mice.
- Statistical evaluation:
Regarding micronucleated PCE and NCE counts and the ratio PCE/NCE at all sample times there were neither biologically relevant nor statistically significant differences (P > 0.05) besides one ex-ception: the analysis of the ratio PCE/NCE showed a significant interaction (P < 0.05) between the factors "sex" and "treatment"; the pair-wise comparisons with the control group indicated a decrease of the ratio for the males in the high dose group 48h after treatment.

Any other information on results incl. tables

MORTALITY (number of deaths/number of animals tested):

Control: Males, 0/15; Females, 0/15

0.5 g/kg bw: Males, 0/15; Females, 1/15

1.0 g/kg bw: Males, 0/15; Females, 2/15

2.0 g/kg bw: Males, 0/15; Females, 1/15

In the 2.0 g/kg bw dose group, animals showed signs of slight to moderate apathy.

MEAN PCE VALUES

Vehicle Control: Males, 0.90 (24 h), 1.10 (48 h), 0.80 (72 h) Females, 1.20 (24 h), 0.80 (48 h), 1.00 (72 h) 

0.5 g/kg bw: Males, 1.30 (24 h), 0.70 (48 h), 0.80 (72 h) Females, 1.12 (24 h), 0.40 (48 h), 0.80 (72 h)

1.0 g/kg bw: Males, 0.60 (24 h), 1.10 (48 h), 0.90 (72 h) Females, 0.88 (24 h), 0.90 (48 h), 0.88 (72 h)

2.0 g/kg bw: Males, 1.50 (24 h), 1.40 (48 h), 1.10 (72 h) Females, 1.10 (24 h), 1.12 (48 h), 0.90 (72 h)

Positive Control: Males, 35.80 (24 h)  Females, 33.00 (24 h)

MEAN NCE VALUES

Vehicle Control: Males, 0.80 (24 h), 0.80 (48 h), 1.00 (72 h) Females, 0.80 (24 h), 0.80 (48 h), 1.40 (72 h) 

0.5 g/kg bw: Males, 1.60 (24 h), 0.80 (48 h), 0.60 (72 h) Females, 0.75 (24 h), 0.60 (48 h), 0.60 (72 h)

1.0 g/kg bw: Males, 1.20 (24 h), 1.40 (48 h), 1.40 (72 h) Females, 1.25 (24 h), 0.40 (48 h), 0.50 (72 h)

2.0 g/kg bw: Males, 1.00 (24 h), 1.20 (48 h), 1.00 (72 h) Females, 0.60 (24 h), 0.50 (48 h), 0.80 (72 h)

Positive Control: Males, 2.60 (24 h)  Females, 2.60 (24 h)

MEAN RATIO PCE/NCE VALUES

Vehicle Control: Males, 1.14 (24 h), 0.96 (48 h), 0.95 (72 h) Females, 1.07 (24 h), 1.12 (48 h), 1.08 (72 h) 

0.5 g/kg bw: Males, 1.08 (24 h), 1.07 (48 h), 0.99 (72 h) Females, 1.12 (24 h), 1.04 (48 h), 1.20 (72 h)

1.0 g/kg bw: Males, 1.00 (24 h), 1.01 (48 h), 1.02 (72 h) Females, 1.09 (24 h), 1.06 (48 h), 1.11 (72 h)

2.0 g/kg bw: Males, 1.03 (24 h), 0.76* (48 h), 1.07 (72 h) Females, 0.95 (24 h), 1.14 (48 h), 1.05 (72 h) * results were significant (i.e., P<0.05) compared to vehicle control

Positive control: Males, 0.81 (24 h)  Females, 0.79 (24 h)

PCE and NCE counts and the ratio PCE/NCE were not biologically relevant,  nor were there statistically significant differences (i.e, P>0.05) at all  sample times, with one exception.  The PCE/NCE ratio showed a significant  interaction between the factors "sex" and "treatment." Pair-wise comparisons with the control group indicated a decrease of the ratio for males in the 2.0 g/kg bw dose group, 48 hrs after treatment. Under the experimental conditions, octyltin trichloride failed to produce any increase in the number of micronucleated polychromatic or normochromatic erythrocytes in male and female mice.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
From the results obtained, it was concluded that ZK 26.384 failed to produce any increase in the number of micronucleated polychromatic or normochromatic erythrocytes in male and female mice.

Executive summary:

15 male and 15 female NMRI mice per group were given once by gavage 0.5, 1.0 or 2.0 g ZK 26.384/kg body weight. Control animals (15 males and 15 females) were given the vehicle at 10 ml/kg in the same manner. Triaziquone (0.15 mg/kg; single i.p. treatment) served as positive control (5 males, 5 females).

5 males and 5 females from the negative control and the ZK 26.384 groups were killed at intervals of 24, 48 and 72 hours after treatment. The positive control animals were killed 24 hours after treatment. Femur bone marrow smears were prepared and stained using May-Gruenwald and Giemsa solutions.

The coded slides were examined for the presence of micronuclei in 2000 polychromatic and 1000 normochromatic erythrocytes and for the ratio of polychromatic to normochromatic erythrocytes.

One day after application overall 4 females died out of all three dose groups. The micronucleated cell counts obtained with ZK 26.384 were comparable with the concurrent control values. Bone marrow depression was observed once at the high dose group for the males at the second sample time.

The positive control compound triaziquone proved to be toxic and gave the expected increase in the micronucleated cell counts.

Conclusion

Evaluation of the data indicates that ZK 26.384 failed to show any evidence of mutagenic potential, when administered by gavage up to the toxic dose level of 2.0 g/kg in the mouse micronucleus test.

Triaziquone, the positive reference gave the expected mutagenic response.