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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: valid without restriction; GLP guideline study conducted by a method similar to OECD Guideline 474.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Test substance:
-Test substance (as cited in report): 2, 2, 4-Trimethyl-1, 3-Pentanediol Monoisobutyrate (Texanol; Texanol Ester-Alcohol)
-Physical description: Colorless liquid
-Source of test material: Eastman Chemical Company
-Purity: 98.8% at start of study
-Purity at end of dosing: 98.7%
-Stability: Based on purity analysis at start and end of study, test material is considered stable.
-Analytical laboratory: Purity/stability analysis performed by: Chemical Quality Services Division, Eastman Kodak Company


Key study dates:
-Experimental initiation date (LD50/3 phase): May 25, 1991
-Experimental initiation date (Micronucleus phase): June 25, 1991
-Experimental completion date: August 27, 1991

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals and environmental conditions:
-Strain/Source: Swiss CD-1, Crl:CD-1 (ICR)BR [Charles River Laboratories, Inc., St. Constant, Canada (LD50/3 phase) and Portage, MI (micronucleus phase)]
-Age (LD50/3 phase): Males, 76 days; Females, 68 days
-Age (micronucleus phase): Males, 66 days; Females, 58 days
-Body weight (LD50/3 phase): 26.7-31.8 g
-Body weight (micronucleus phase): 24.5-36.1 g
-Acclimatization: 5 days
-Housing: one or two per cage
-Temperature (°F) 72 ±3
-Humidity (%): 50 ± 20
-Photoperiod: 12 hours
-Feed: Agway Prolab RMH 3000 pellets ad libitum
-Water: Rochester, NY local municipality water ad libitum
-Randomization: randomly assigned to study groups based on individual body weights
-Identification: uniquely identified by ear tag

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Distilled water
Details on exposure:
LD50/3 phase:
A dose of 2000 mg/kg bw undiluted test substance was administered by gavage to three male and three female mice. Mice were observed for 24 hours.

Micronucleus phase:
Mice were administered the test substance via oral gavage at dose levels of 0, 200, 1000, and 2000 mg/kg bw. The negative control group was administered 2.11 mL/kg bw of distilled water while the positive control group received 80 mg/kg bw cyclophosphamide. Twenty-four hours after Texanol or negative control administration, one half of the animals were euthanized, femurs removed, and bone marrow smears prepared. Positive control animals were all euthanized at 24 hours. The remaining animals were euthanized 48 hours after test substance or negative control administration, femurs removed, and bone marrow smears prepared.
Duration of treatment / exposure:
Once
Frequency of treatment:
Once
Post exposure period:
24 (Texanol, negative control, and cyclophosphamide) and 48 hours (Texanol and negative control)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2000 mg/kg bw Texanol
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw Texanol
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg bw Texanol
Basis:
actual ingested
No. of animals per sex per dose:
5 animals/sex/dose:harvest time group
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control:
-Cyclophosphamide (Aldrich Chemical Co)
-Lot number: CW-05609LT
-Purity per supplier: >98%
-A sample analyzed by hplc before study initiation was found to be free of detectable impurities.

Examinations

Tissues and cell types examined:
Slides were scored for micronuclei and the relative numbers of polychromatic (PCE) and normochromatic (NCE) cells. One thousand polychromatic cells per animal were routinely scored.
Details of tissue and slide preparation:
At harvest, the mice were euthanized with carbon dioxide and the adhering soft tissue and epiphyses of both femurs were removed. The marrow was flushed from the femurs, transferred to centrifuge tubes containing 2 mL fetal calf serum (1 tube/animal), centrifuged to create a pellet, supernatant removed, and the cells were resuspended. The suspension was spread on glass microscope slides and air dried.

The slides were then fixed in methanol, stained in Wright’s-Giemsa stain, rinsed in deionized water, allowed to air dry, and coverslipped using mounting medium. The slides were labelled in such a manner that the group was unknown to the examiner.
Evaluation criteria:
General:
The criteria for identification of micronuclei were those of Schmid (1976). The unit of scoring was the micronucleated cell, not the micronucleus; thus the occasional cell with more than one micronucleus was counted as one micronucleated PCE.

Interpretation:
A positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant increase in micronucleated PCEs for at least one dose level. A test substance that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative.

Bone marrow depression:
Bone marrow depression was defined as a statistically significant decrease in the percent PCE ([PCE/PCE + NCE] X 100) in treated animals compared to the corresponding negative controls.
Statistics:
The unit of scoring and statistical analysis was the micronucleated cell, not the micronucleus. The data were examined using plots, descriptive statistics, rank transformation, analysis of variance, Tukey’s HSD Test and Dunnett’s t-test. Statistical significance was detected using an alpha risk of ≤ 0.05.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
The test substance produced transient signs of acute toxicity in the high-dose female mice.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Immediately after dosing, 2 of 10 high-dose females showed prostration, 4 of 10 exhibited lethargy, 1 of 10 was hypothermic, and another was ataxic. All animals were recovering by the 4-hr observation period, all appeared normal at 24 hours after dosing and remained healthy until the appropriate harvest times. All males, and the low- and mid-dose females appeared normal throughout the study.

The only statistically significant effect was a decrease in the number of micronucleated PCE’s when compared to controls in the 1000 mg/kg bw male mice at the 48 hour harvest time. Both sexes of mice treated with 80 mg/kg bw cyclophosphamide showed statistically significant increases in micronucleated PCEs and a statistically significant bone marrow depression at 24 hours when compared with the negative control.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test substance, 2, 2, 4-trimethyl-1, 3-pentanediol monoisobutyrate (Texanol) did not induce significant increases in micronuclei in bone marrow polychromatic erythrocytes under the conditions of this assay in any dose group at any harvest time.

Based on an absence of genotoxic/mutagenic effects in this study, Texanol® Ester-Alcohol is not classified for “Germ Cell Mutagenicity” according to GHS.
Executive summary:

In an in vivo bone marrow micronucleus assay, groups of male and female CD-1 mice were administered a single dose of 2, 2, 4-trimethyl-1, 3-pentanediol monoisobutyrate (Texanol) by oral gavage at dose levels of 0, 200, 1000, and 2000 mg/kg bw. Animals dosed with the test substance were euthanized at 24 and 48 hours after dosing for extraction of the bone marrow. A distilled water control group was included at each harvest time and a positive control group, dosed with cyclophosphamide at 80 mg/kg bw, was included only at the 24-hr sampling time. At the highest dose tested, Texanol did not induce a statistically significant increase in micronuclei in bone marrow polychromatic erythrocytes or a significant level of bone marrow depression in either male or female mice, at either the 24-hr or 48-hr harvest time when compared with concurrent vehicle controls. The positive control, cyclophosphamide, induced highly significant increases in micronucleated polychromatic erythrocytes in both sexes of mice at 24 hours and significant bone marrow depression when compared with the controls. It is concluded that Texanol is negative in the in vivo mammalian bone marrow micronucleus assay.