Registration Dossier

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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well described study and suitable for assessment.

Data source

Referenceopen allclose all

Reference Type:
secondary source
Title:
Unnamed
Year:
1995
Reference Type:
other: Publication; reference for detailed protocol
Title:
The in Vivo Erythrocyte Micronucleus Test: Measurement at Steady State Increases Assay Efficency and Permits Integration with Toxicity Studies
Author:
MacGregor JT, Wehr CM, Henika PR, Shelby MD
Year:
1990
Bibliographic source:
Fundamental and Applied Toxicology, volume 14, pages 513 - 522

Materials and methods

Principles of method if other than guideline:
Peripheral blood samples were obtained from male and female B6C3Fl mice at the end of the 13-week drinking-water toxicity study. Smears were prepared, fixed, and stained. The frequency of micronuclei in 10,000 normochromatic erythrocytes (NCB) in up to 10 animals per dose group was determined.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: colorless liquid
Details on test material:
- Name of test material (as cited in study report): t-butyl alcohol
- Physical state: clear colorless liquid
- Analytical purity: > 99 %
- Lot/batch No.: F112784
- Stability: bulk chemical is stable for 2 weeks
- Stability under test conditions: The stability of the dose formulations was at least 3 weeks at room temperature when stored in the dark and at least 3 days at room temperature under normal room light.
- Storage condition of test material: it should be protected from light at temperatures up to 60 °C

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Frederick Cancer Research Facility
- Age at study initiation: approximately 6 weeks old
- Housing: single housing
- Diet (e.g. ad libitum): NIH-07 open formula rodent feed; ad libitum
- Water (e.g. ad libitum): deionized water; ad libitum
- Acclimation period/quarantine: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 12-30 °C
- Humidity (%): 31-56 %
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
- Vehicle(s)/solvent(s) used: water

Details on exposure:
REPARATION OF DOSING SOLUTIONS: T-butyl alcohol was mixed with deionized water
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
94-95 consecutive days in the drinking water
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 2.5, 5, 10, 20 or 40 mg/mL in deionized
Basis:
nominal in water
No. of animals per sex per dose:
Male:
0: 8
2.5 mg/mL: 10
5 mg/mL: 10
10 mg/mL: 9
20 mg/mL: 9
40 mg/mL: 3

Female:
0: 9
2.5 mg/mL: 8
5 mg/mL: 10
10 mg/mL: 10
20 mg/mL: 9
40 mg/mL: 5
Control animals:
yes, concurrent vehicle
Positive control(s):
- used positive control: Urethane
- Doses / concentrations: 2,000 ppm

Examinations

Tissues and cell types examined:
Normochromatic erythrocytes
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION: Smears were prepared and fixed in absolute methanol, stained with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y, and coded.

METHOD OF ANALYSIS: Slides were scanned at 630x or 1,000x magnification using a semi-automated image analysis system to determine the micronuclei in 10,000 normochromatic erythrocytes (NCB).
A detailled discussion of this assay can be found in MacGregor et al., 1990.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No increase in the frequency of micronucleated NCEs was observed. No effect on the percentage of PCEs was noted.

Any other information on results incl. tables

Males

Dose (mg/mL)

Micronucleated NCE cells (%)

PCE (%)

Number of Mice

 

0

0.09 +/- 0.01

0.86 +/- 0.14

8

 

2.5

0.10 +/- 0.01

1.03 +/- 0.07

10

 

5

0.07 +/- 0.01

0.92 +/- 0.16

10

 

10

0.09 +/- 0.02

0.68 +/- 0.08

9

 

20

0.08 +/- 0.01

0.87 +/- 0.10

9

 

40

0.06 +/- 0.03

0.52 +/- 0.25

3

 

 

 

 

 

Urethane

2

1.95 +/- 0.07

1.79 +/- 0.07

3

 

 

 

 

 

Females

Dose (mg/mL)

Micronucleated NCE cells (%)

PCE (%)

Number of Mice

 

0

0.06 +/- 0.01

0.88 +/- 0.13

9

 

2.5

0.04 +/- 0.01

0.68 +/- 0.10

8

 

5

0.05 +/- 0.01

0.77 +/- 0.16

10

 

10

0.05 +/- 0.01

0.88 +/- 0.09

10

 

20

0.07 +/- 0.01

0.94 +/- 0.08

9

 

40

0.07 +/- 0.01

0.81 +/- 0.18

5

Table 3: Frequency of Micronuclei in Mouse Peripheral Blood Erythrocytes Following Treatment with t-Butyl Alcohol in Drinking Water for 13 Weeks.

Applicant's summary and conclusion