Isooctyl acetate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 December 1993 - 30 March 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vivo mammalian bone marrow micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Isooctyl acetate

EC Number:

250-701-2

EC Name:

Isooctyl acetate

Cas Number:

31565-19-2

Molecular formula:

C10H20O2

IUPAC Name:

Acetic acid, isooctyl ester

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Exxon Chemical Company / Lot Number: 111991
- Expiration date of the lot/batch: 31 October 1998
- Purity test date: Considered 100% pure

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: The test substance was weighed out and on the day of dosing, mixed with the carrier to provide stock solutions such that individual animal dose volumes did not exceed 1.0 ml/100 grams body weight.

Test animals

Species:

mouse

Strain:

other: CD-I (VAF/Plus)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Portage, Michigan
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: 20 to 31 grams
- Assigned to test groups randomly: Yes
- Housing: Single housed in suspended stainless steel and wire mesh cages with absorbent paper below.
- Diet (e.g. ad libitum): Purina Certified Rodent 5002 Chow (pellets) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Fourteen days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-25°C
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): Approximately 12 hours light/dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was weighed out and on the day of dosing, mixed with the carrier to provide stock solutions such that individual animal dose volumes did not exceed 1.0 ml/100 grams body weight. The test substance was assumed to be 100% pure for purposes of testing and there was no attempt made to correct for the actual purity of the test substance.

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

625 mg/kg bw (total dose)

Doses / concentrations 2

Dose / conc.:

1 250 mg/kg bw (total dose)

Doses / concentrations 3

Dose / conc.:

2 500 mg/kg bw (total dose)

No. of animals per sex per dose:

10

Control animals:

yes

yes, concurrent vehicle

other: Positive control: Cyclophosphamide

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): The positive control was considered to be stable under the conditions of the assay in that it performed in a manner consistent with data from previous studies.
- Route of administration: Oral gavage
- Doses / concentrations: 40mg/kg

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A rangefinding study was performed using test concentrations 1.25, 2.5 and 5.0 g/kg. Three of the four animals from test concentration 5.0 g/kg died prior to sacrifice. The test concentration of 2.5 g/kg appeared to be the maximum tolerated dose. Therefore test concentrations of 0.625, 1.25 and 2.5 g/kg were tested in the micronucleus assay.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Animals were sacrificed at approximately 24, 48, and 72 hours after dose administration.

DETAILS OF SLIDE PREPARATION: Immediately after sacrificing both femurs were removed. The bone marrows were aspirated, flushed in fetal bovine serum and centrifuged. After decanting the supernatant, the cell pellet was resuspended in any remaining supernatant. Smears, two slides per animal, were prepared. Slides were stained using Acridine Orange and were wet mounted.

METHOD OF ANALYSIS: 1000 polychromatic erythrocytes from each animal were examined for the presence of micronuclei. The percent of polychromatic erythrocytes in the total population of erythrocytes was determined for each animal by counting a total of 1000 polychromatic and normochromatic erythrocytes. Polychromatic erythrocytes stain fluorescent red/orange, normochromatic erythrocytes are unstained or stain dull green and micronuclei stain fluorescent bright yellow. Additional criteria for scoring micronuclei are a circular appearance and a diameter between 1/20 and 1/5 of the cell's diameter

Evaluation criteria:

The criteria for the test substance to be considered as inducing a positive response when compared to the carrier control are a dose-related statistical increase in the mean number of micronucleated polychromatic erythrocytes, including at least one dose point that is statistically different from the mean number of micronucleated polychromatic erythrocytes of the carrier control. This value also must be outside the normal range of the mean number of micronucleated polychromatic erythrocytes of the carrier control; or: at least two dose points that are statistically different from the mean number of micronucleated polychromatic erythrocytes of the carrier control and greater than the normal range of the mean number of micronucleated polychromatic erythrocytes of the carrier control. A dose point is considered negative if the mean value for a statistically significant increase in micronuclei is within the normal range of the carrier control (0-4 micronuclei per 1000 polychromatic erythrocytes). The assay will be considered inconclusive if a single dose is positive and there is no dose response.

Statistics:

Statistical analysis included calculation of means and standard deviations of the micronuclei data and a test of equality of group means by a standard one way analysis of variance at each time period. When the ANOVA was significant, comparisons of carrier control to dosed group means were made according to Duncan's Multiple Range Test. A standard regression analysis was performed to test for a dose response. Residuals from the ANOVA were analyzed for normality by Wilk's criterion. The residuals were normally distributed (values were greater than 0.01 level of significance in more than 25% of the analyses). Therefore, nonparametric analysis was not performed. Sexes were analyzed separately.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

A statistically significant increase in the mean number of micronucleated polychromatic erythrocytes was not observed for any of the concentrations tested. Cytotoxicity, a dose related decrease in the percentage of polychromatic erythrocytes as compared to the carrier control, was observed at the 48 hour sampling time in both sexes (regression coefficient P<0.01). Test concentrations of 1.2 g/kg and 2.5 g/kg were significantly different from the carrier control for both sexes (P<0.05 for the females, P<0.01 for the males). A dose related decrease in the percentage of polychromatic erythrocytes was observed for the females at the 24 hour and 72 hour sampling times and for the males at the 24 hour sampling time (regression coefficient at P<0.05). However, none of the dose groups were statistically different from the control.

 

The positive control induced a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes (P<0.01) which indicates that the positive control is clastogenic and is responding in an appropriate manner.

Carrier control values for the mean percent of polychromatic erythrocytes and for the mean percent of micronucleated polychromatic erythrocytes responded in an appropriate manner.

Applicant's summary and conclusion

Conclusions:

The test substance did not induce a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes in the bone marrow cells of CD-I mice. Therefore, Exxate 800 is not considered mutagenic under the conditions of this assay.

Executive summary:

The micronucleus assay is a short term test to evaluate the clastogenic (i.e., chromosome breaking) potential of test substances (Schmid, 1976). Evidence of chromosome breakage or nondisjunction can be readily detected as micronucleated polychromatic erythrocytes. Positive test results are considered to provide presumptive evidence of mutagenic potential in mammals, (Heddle et al., 1983).

This study was conducted in compliance with the U.S. EPA, Federal Insecticide, Fungicide and Rodenticide Act (FIFRA); Good Laboratory Practice Standards, 40 CFR Part 160, 1989 and the U.S. EPA, Toxic Substance and Control Act (TSCA) Test Guidelines 40 CFR 798.5395, 1985 to determine if Exxate 800 (MRD-93.688) was capable of inducing an increase in the percentage of micronucleated polychromatic erythrocytes in the bone marrow of CD-I mice.

The test substance was administered by oral gavage as a single dose using corn oil as the carrier. A rangefinding study was performed using test concentrations 1.25, 2.5 and 5.0 g/kg. Three of the four animals from test concentration 5.0 g/kg died prior to sacrifice. An increase in micronuclei formation was not observed for any of the surviving animals at any of the concentrations tested.

The test concentration of 2.5 g/kg appeared to be the maximum tolerated dose. Therefore, test concentrations of 0.625, 1.25 and 2.5 g/kg were tested in the micronucleus assay. Bone marrow samples were collected and evaluated for micronucleus formation 24, 48 and 72 hours after dosing.

An increase in micronuclei formation was not observed at any of the test concentrations. Cytotoxicity, a decrease in the percentage of polychromatic erythrocytes as compared to the carrier control, was observed at the 48 hour sampling time for both sexes (regression coefficient P< 0.01). Test concentrations of 1.25 and 2.5 g/kg were significantly different from the carrier control for both sexes (P<0.05 for the females , P<-.01 for the males).

Under the conditions of this test, MRD-93-688 (Exxate 800), did not produce an increase in micronuclei formation in the bone marrow of CD-I mice. The test substance did induce a decrease in polychromatic erythrocytes as compared to the carrier control, which is a measure of cytotoxicity.

The assay contained positive (cyclophosphamide) and negative (carrier) controls, both which responded in an appropriate manner.