Registration Dossier

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:
13 weeks
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The exposure portion of the study was conducted according to NTP standard study and GLP guidelines for a 13-week drinking water study. The in vivo erythrocyte micronucleus test was conducted according to methods similar to those outlined in OPPTS 870.5395 and EU B.12 guidelines for the conduct of an in vivo mammalian erythrocyte micronucleus test. MacGregor et al. (1990), which discusses the merits of performing the analysis after multiple exposures to obtain maximum micronucleus frequency or a steady state rather than the more traditional method which uses a single exposure or two exposures at a 24-hr interval, was also cited.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: NTP standard guidelines for subchronic drinking water study
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
not specified
Qualifier:
equivalent or similar to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-methylpropan-2-ol
EC Number:
200-889-7
EC Name:
2-methylpropan-2-ol
Cas Number:
75-65-0
Molecular formula:
C4H10O
IUPAC Name:
2-methylpropan-2-ol
Constituent 2
Reference substance name:
tertiary butyl alcohol
IUPAC Name:
tertiary butyl alcohol
Details on test material:
-Name of test material (as cited in study report): t-butyl alcohol
-Source of test material: FBC Chemical Corporation (Lancaster, NY)
-Analytical laboratory: Identity, purity and stability analyses were conducted by the analytical chemistry laboratory, Midwest Research Institute (Kansas City, MO)
-Analysis method for identity confirmation: infrared, UV/Vis, NMR
-Analytical purity by GC: > 99%
-Lot No.: F112784
-Stability under test conditions: Stability studies of the bulk chemical were performed using gas chromatography. These studies indicated that tertiary butyl alcohol was stable as a bulk chemical for two weeks when stored protected from light at temperatures up to 60 °C. Stability was monitored during the 13-week study using gas chromatography. No degradation of the bulk chemical was detected.
-Storage condition of test material: At room temperature in amber glass bottles with Teflon-lined lids.

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 (Frederick, MD)
-Age at receipt: approx. 4 weeks; animals were held for 13 days before study initiation
-Health prior to study initiation: no ectoparasites, endoparasites or gross abnormalities
-Health at study termination: serology screening did not indicate the presence of any viral pathogens
-Age at study initiation: approx. 6 weeks
-Average age at necropsy: 19 weeks
-Method of Animal Identification: toe clip
-Method of Animal Distribution: Animals randomized from weight classes into cage groups using a random numbers table; cages randomized into test groups using a random numbers table
-Housing: Animals were individually housed in solid-bottom, polycarbonate cages (Lab Products, Inc., Maywood, NJ) lined with Beta-Chips® heat-treated hardwood-chip bedding (Northeastern Products, Inc., Warrensburg, NY). Cages were changed weekly and rotated every two weeks. Bedding was changed weekly. Cages were suspended on stainless steel racks (Lab Products, Inc., Maywood, NJ); racks were rotated every 2 weeks.
-Diet: NIH-07 Open Formula Meal Diet (Zeigler Brothers, Inc., Gardners, PA) was available ad libitum. Food was changed weekly.
-Water: city water (filtered and deionized) was available ad libitum as plain or dosed

ENVIRONMENTAL CONDITIONS:
-Temperature (°C): 18-24
-Relative Humidity: 32% to 58%
-Air changes per hour: minimum of 10 changes/hr
-Photoperiod (hrs dark/ hrs light): 12 hours light/dark

IN-LIFE DATES:
-Date of First Dose: 10 December 1985
-Date of Last Dose: 13-14 March 1986
-Duration of Dosing: 94-95 days
-Date of Necropsy: 13-15 March 1986

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
deinonized water
Details on exposure:
The dose formulations were prepared by mixing tertiary butyl alcohol with deionized water for a concentration of 0, 2.5, 5, 10, 20 or 40 mg/ mL (0, 0.25, 0.5, 1.0, 2.0, and 4.0%). Formulations were stored in glass bottles at room temperature for up to 2 weeks. Animals were allowed ad libitum access to drinking water (plain or dosed) for 94 to 95 days.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
ad libitum in the drinking water
Post exposure period:
None
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
In 13-week study, identified as 2.5 mg/mL; 0.25% targeted (delivered average daily dose of 350 mg tertiary butyl alcohol/kg bw/day for males and 500 mg/kg bw/day for females). In micronucleus phase, reported as 3000 ppm.
Basis:
nominal in water
Remarks:
Doses / Concentrations:
In 13-week study, identified as 5.0 mg/mL; 0.5% targeted (delivered average daily dose of 640 mg tertiary butyl alcohol/kg bw/day for males and 820 mg/kg bw/day for females). In micronucleus phase, reported as 5000 ppm.
Basis:
nominal in water
Remarks:
Doses / Concentrations:
In 13-week study, identified as 10 mg/mL; 1.0% targeted (delivered average daily dose of 1590 mg tertiary butyl alcohol/kg bw/day for males and 1660 mg/kg bw/day for females). In micronucleus phase, reported as 10000 ppm.
Basis:
nominal in water
Remarks:
Doses / Concentrations:
In 13-week study, identified as 20 mg/mL; 2.0% targeted (delivered average daily dose of 3940 mg tertiary butyl alcohol/kg bw/day for males and 6430 mg/kg bw/day for females). In micronucleus phase, reported as 20000 ppm.
Basis:
nominal in water
Remarks:
Doses / Concentrations:
In 13-week study, identified as 40 mg/mL; 4.0% targeted (delivered average daily dose of 8210 mg tertiary butyl alcohol/kg bw/day for males and 11620 mg/kg bw/day for females). In micronucleus phase, reported as 40000 ppm.
Basis:
nominal in water
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control(s):
Three male mice were exposed separately to urethane for the micronucleus phase and were not part of the NTP study.

Examinations

Tissues and cell types examined:
For the micronucleus phase, peripheral blood samples were obtained from surviving male and female mice at the end of the 13-week toxicity study. Blood was analyzed for percentage of polychromatic erythrocyes (PCEs) among the total erythrocyte population and frequency of micronuclei in 10000 normochromatic erythrocytes.
Details of tissue and slide preparation:
Once blood was drawn, smears were immediately prepared and fixed in absolute methanol, stained with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y described by MacGregor et al., (1983) and coded.

Reference cited: MacGregor et al., 1983. A simple fluorescent staining procedure for micronuclei and RNA in erythrocytes using Hoechst 33258 and pyronin Y. Mutat. Res. 120, 269-275.
Evaluation criteria:
Slides were scanned at 630x or 1000x magnification using a semi-automated image analysis sytem to determine the frequency of micronuclei in 10,000 normochromatic erythrocytes (NCEs) in up to 10 animals per dose group. The criteria of Schmid (1976) were used to define micronuclei, with the additional requirement that the micronuclei exhibit the characteristic fluorescent emissions of DNA (blue with 360 nm and orange with 510 nm UV illumination); the minimum size limit was approximately 1/20 the diamter of the NCE cell. In addition, the percentage of polychromatic erythrocytes (PCEs) among the total erythrocyte population was determined.

Reference cited: Schmid W, 1976. The micronucleus test for cytogenetic analysis. In Chemical Mutagens: Principles and Methods for their Detection (A. Hollaender, Ed.), Vol. 4, pp. 31-53. Plenum Press, New York.
Statistics:
Data for percent micronucleated NCE cells and percent PCE were presented as mean ± standard error.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Although various signs of toxicity were observed in both sexes during the 13-week exposure phase and at necropsy, no effect on the percentage of PCEs in the total erythrocyte population was noted, an idication that tertiary butyl alcohol was not toxic to bone marrow cells.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Tertiary butyl alcohol did not cause cytogenetic damage resulting in the formation of micronuclei in mouse peripheral erythrocytes when administered to male and female mice ad libitum in drinking water at concentrations up to 4% for 13 weeks.

Based on an absence of genotoxic/mutagenic effects in this study, tertiary butyl alcohol is not classifiable for Germ Cell Mutagenicity according to GHS.
Executive summary:

In a mammalian erythrocyte micronucleus test, groups of 10 male and 10 female mice were exposed to tertiary butyl alcohol ad libitum in drinking water for 13 weeks at dose levels of 0, 0.25, 0.5, 1.0, 2.0 and 4.0%. The positive control induced the appropriate response.  In vivo, no statistically significant increase in the frequency of micronucleated NCEs was observed in male or female mice administered tertiary butyl alcohol in drinking water for 13 weeks.  In addition, no effect on the percentage of PCEs in the total erythrocyte population was noted, an indication that tertiary butyl alcohol was not toxic to bone marrow cells.