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Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
other: re-evaluation of expermental result
Adequacy of study:
supporting study
Study period:
12 May 1986- 12 Nov 1986
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature

Data source

Reference
Reference Type:
other company data
Title:
Unnamed
Year:
1986
Report date:
1986

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
A micronucleus test with TBTO was performed by the Institute of Occupational Health/Helsinki, Finland. 10 male and 10 female Balb/c mice per group were given once by gavage 30 or 60 mg TBTO/kg bw as a solutoin in olive oil. Control animals (10 males and 10 females) were given the vehicle at 20 mL/kg in the same manner. Cyclophosphamide (15 mg/kg; single i.p. treatment) served as a positive control.
5 males and 5 females from the negative and positive control and the TBTO groups were killed at intervals of 30 and 48 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 1000 PCE and 1000 NCE and for the ratio of PCE/NCE.
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
tri-n-butyltin oxide
IUPAC Name:
tri-n-butyltin oxide
Constituent 2
Reference substance name:
ZK 21.955
IUPAC Name:
ZK 21.955
Details on test material:
no data

Test animals

Species:
mouse
Strain:
Balb/c
Sex:
male/female
Details on test animals or test system and environmental conditions:
no data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
olive oil
Details on exposure:
30 or 60 mg/kg
Duration of treatment / exposure:
single exposure
Frequency of treatment:
single exposure
Post exposure period:
Animals were sacrificed at 30 and 48 h after treatment
Doses / concentrationsopen allclose all
Dose / conc.:
30 other: mg/kg (nominal)
Dose / conc.:
60 other: mg/kg (nominal)
No. of animals per sex per dose:
10/sex treated animals
5/sex negative and positve controls
Control animals:
yes
Positive control(s):
Cyclophsophamide 15 mg/kg single ip treatment

Examinations

Tissues and cell types examined:
erythrocytes from femur bone marrow smears
Details of tissue and slide preparation:
Femur bone marrow smears were prepared and stained using May-Gruenwald and Giemsa solutions
Evaluation criteria:
Finish study: presence of micronuclei in 1000 PCE and 1000 NCE (polychromatic erythrocytes/normochromatic erythrocytes)
Re-evaluation: presence of micronuclei in 1000 PCE and 1000 NCE (with oil immersion high power magnification; frequency of PCE was calculated on basis of 1000 NCE
Statistics:
Re evaluation: statistical anlayis was conducted on the following variables: P1 (proportion of micronucleated PCE), P2 (proportion of micronucleated NCE) and P3: ratio of PCE/NCE. P1 and p2 were transformed (arc sin sqare root of Pi). Analyses conducted for spearate sample times. ANOVA with factors sex and treatment was performed to assess difference tbetween positive and negative contreols with pooled values for both sexes; thereafter the postive contorl group was excluded fromfurther analysis.
Comparison between control fo each treatment group were conducted separately for the sexes. Compariosns were performed with values pooled for both sexes if no differece was observed. Pairwise comparisons were performed with one-sided t-tests (increase of P1 and P2, decrease of P3), using the error estimate of the ANVOA table. The test levels were always alpha = 0.05, (least significant differences test, LSD). Arithmetic means and std deviations included.

Results and discussion

Any other information on results incl. tables

Regarding the first sample time there were no significant findings. Regarding the second sample time, a significant treatment effect was revealed for both dose levels with respect to micronucleated PCE in females. The micronucleated NCE-counts showed - pooled for both sexes - a significant increase at the high dose level. The ratio PCE/NCE decreased significantly in males at both dose levels. The positive control group was significantly different from the control group with respect to the micronucleated PCE counts at the first sample time and the micronucleated NCE counts at both sample times. 

The slight but statistically significant increases of the micronucleated PCE counts in females at both TBTO groups (3,2,3,2,1 and 1,1,2,1,0) versus the concurrent negative control (0,1,0,0,1) at the second sample time was biologically not relevant: the concurrent control values in females were unusually low compared to those of the first sample time (PCE: 1,1,2,0; NCE: 1,1,3,0) or the control values of the males (PCE: 2,1,2,0,1 and 0,2,2,1,1; NCE: 1,0,1,1,2 and 1,1,2,0,3) and moreover there was no dose response relationship at all, on the contrary, there was a decrease in the mean from 2.2 (30 mg/kg) to 1.0 (60 mg/kg) with no significant evidence of bone marrow depression. The same holds true for the increase in micronucleated NCE. The fact that there was no significant increase in micronucleated PCE in males at the second sample time as observed by the Finnish study, but on the contrary in female mice, illustrates clearly the variability of micronucleus test data obtained from the same study, if only 1000 PCE per animal were scored. According to a very recent collaborative study on sex difference in the mouse micronucleus test (Mutation Res. 172, 151-163, 1986) all twenty clastogens tested induced micronuclei in both sexes. 

Applicant's summary and conclusion

Conclusions:
The reevaluation of the slides and statistical analysis indicates TBTO failed to show evidence of mutagenic potential when administered by gavage up to 60 mg/kg in the mouse micronucleus test.
Executive summary:

This study was a re-evaluation of the micronucleus test completed by the Finnish Institute of Occupational Health (1984). The slides were requested for recounting because a similar study completed by Schering (dated 29 Aug1983) did not show any evidence of mutagenic activity. The Finnish study reported mutagenic activity; however, both the spontaneous background rates and mean ratio polychromatic erythorictyes/normochromatic erythrocytes ((PCE/NCE) were unusually high. Evaluation of the data after counting of 1000 PCE and 1000 NCE per animal indicates that TBTO failed to show evidence of mutagenic potential, when administered by gavage up to 60 mg/kg, in the mouse micronucleus test. Cyclophosphamide, the positive reference gave the expected mutagenic response. The result indicates that the most likely explanation for the difference to the outcome of the Finnish report is due to the limited PCE sample size (see also Ashby and Mohammed, Mutation Res. 164, 217-235, 1986). The result of the re-evaluation is in agreement with the outcome of an earlier study (Schering, Exp, Toxicology, Report No. IC 5/83, dated Aug. 29, 1983).