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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-04-24 to 1989-04-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study (OECD 474)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Tributylamine
EC Number:
203-058-7
EC Name:
Tributylamine
Cas Number:
102-82-9
Molecular formula:
C12H27N
IUPAC Name:
N,N-dibutylbutan-1-amine
Details on test material:
- Name of test material (as cited in study report): Tributylamine
- Physical state: clear up to light yellow liquid
- Analytical purity: 99.3 %
- Purity test date: 1989-03-08
- Lot/batch No.: 470811

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG, Kastengrund, SPF breeding colony
- Age at study initiation: 7 weeks
- Weight at study initiation: males: 28.9 (26-34) g; females: 24.1 (22-28) g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Housing: groups of 5 animals in Macrolon cages
- Diet (ad libitum): Altromin 1324, altromin GmbH, Lage/Lippe, Germany
- Water (ad libitum): tap water
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
sesame oil
Duration of treatment / exposure:
single exposure
Frequency of treatment:
single exposure
Post exposure period:
animals were sacrificed 24, 48 and 72 h post exposure
Doses / concentrations
Remarks:
Doses / Concentrations:
150 mg/kg bw (single dose) in sesame oil (10 mL/kg bw)
Basis:
nominal conc.
No. of animals per sex per dose:
5 per sex
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide, dose 50 mg/kg bw

Examinations

Tissues and cell types examined:
polychromatic erythrocytes from femoral bone marrow
Details of tissue and slide preparation:
For each animal, about 3 ml foetal bovine serum was poured into a centrifuge tube. Both femora were removed and the bones freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at 1200 rpm and almost all the supernatant discarded. One drop of the thoroughly mixed sediment was smeared on a cleaned slide, identified by project code and animal number and air-dried for about 24 hours.

Staininq procedure
- 5 minutes in methanol
- 3 minutes in May-Grünwalds solution
- 2 minutes in May-Grünwalds solution diluted 1:1 with distilled water
- brief rinsing twice in distilled water
- 10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution, pH 7.2 (Weise)
- rinsing in distilled water
- drying
- coating with Entellan
Evaluation criteria:
1000 polychromatic erythrocytes were examined for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. As a control measure 1000 mature erythrocytes were also counted and examined for micronuclei. In addition, the ratio of polychromatic to normochromatic erythrocytes was determined. All bone marrow smears for evaluation are coded to ensure that the group to which they belonged remains unknown to the investigator. The numbers of polychromatic erythrocytes with micronuclei occurring in the 1000 polychromatic erythrocytes counted, and the number of normocytes with micronuclei occurring in the 1000 normocytes counted, were evaluated statistically.

The results of the treatment groups (test substance) in the micronucleus test at each dose and killing time were compared with corresponding control values. The ratio of polychromatic to normochromatic erythrocytes was also evaluated statistically. Actual data were also compared with historical controls.
Statistics:
Statistical analysis was performed using the paired, one-sided or two-sided Wilcoxon test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
increased as well as reduced spontaneous activity, reversible within 5 h post exposure
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
The incidence of micronucleated polychromatic erythrocytes of the animals treated with tributylamine was within the normal range of the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment with Tributylamin and was statistically not different from the control values.
Cyclophosphamide induced a marked statistically significant increase in the number of polychromatic cells with micronuclei in both males and females, indicating.the sensitivity of the system. The ratio of polychromatic erythrocytes to normocytes was not changed to a significant extend.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the test substance was not mutagenic in the micronucleus test in mice.
Executive summary:

The test compound (purity 99.3%) was administered orally by gavage to male and female NMRI mice (5 per sex). Single doses of 150 mg were tested and the animals were sacrificed 24, 48 and 72 h post exposure. Cyclophosphamide was used as positiv control substance (50 mg/kg bw).

The incidence of micronucleated polychromatic erythrocytes of the animals treated with the test substance was within the normal range of the negative control. The number of normochromatic erythrocytes containing micronuclei was not increased. The ratio of polychromatic/normochromatic erythrocytes in both male and female animals remained unaffected by the treatment and was statistically not different from the control values. The positive control substance induced a marked statistically significant increase in the number of polychromatic cells with micronuclei, indicating.the sensitivity of the system. The test substance did not induce micronuclei in vivo. (Hoechst, 1989).

This study, performed according to OECD guideline 474, was judged ro be reliable (RL1) and selected as key study.