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: GLP guideline study conducted in compliance with OECD Guideline 474, US EPA Health Effects Guideline OPPTS 870.5395, and the EU Method B12.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Identity (according to report): tert-Butyl acetate
- Appearance: Clear, colorless liquid
- Storage Conditions: Room temperature
- Batch Number: 1031267
- Expiry date: April 2001
- Purity: ≥ 95%
- Date received: 30 March 1998
- Date of Dosing: 3 May 2000

Test animals

Species:
rat
Strain:
other: Specific Pathogen Free Sprague-Dawley Hsd/Ola outbreed albino rats
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS:
-Strain: Specific Pathogen Free Sprague-Dawley Hsd/Ola outbred albino rats
-Source: Harlan Olac UK Ltd, Bicester, Oxon, England
-Sex: male/female
-Weight at receipt: 140 - 149 g
-Age at receipt: no data
-Acclimation period: minimum of 5 days
-Housing: The animals were housed and dosed within one room.. Animals were housed in groups, with the sexes separated, in suspended stainless steel cages equipped with solid sides and wire grid front, back and floor. Cage papers were changed daily. Cages were suspended on moveable batteries. The control groups (Groups 1 and 5) were housed on one battery and the test groups on a separate battery. Animals in the positive control group were housed in the same conditions except that they were held in solid plastic cages with stainless steel lids with softwood-chip bedding. The animals within each dosage group were kept on cage batteries within the same room in which the exposure took place.
-Diet: pelleted expanded rat and mouse No. 1 maintenance diet (SQC grade obtained from Special Diets Services Ltd., Witham, Essex, UK), ad libitum except during the period of inhalation exposure
-Water: tap water, ad libitum except during the period of inhalation exposure
-Method of animal identification: coding on tails
-Method of animal distribution: randomly assigned to groups

ENVIRONMENTAL CONDITIONS:
-Temperature (°C): 19.5 - 20
-Humidity (%): 45.5 - 61
-Photoperiod: 12 hours artificial light in each 24-hour period
-Air Exchanges: at least 12 room-air changes per hour

Administration / exposure

Route of administration:
other: Inhalation: nose-only
Vehicle:
clean-air
Details on exposure:
Groups of male and female rats were exposed by inhalation to target concentrations of 100, 400 and 1600 ppm tertiary butyl acetate for 6 hours while the negative control group was exposed to clean-air only. Analytical concentrations for the three exposure groups were determined to be 93, 451 and 2044 ppm, respectively. The positive control group was dosed orally, by gastric intubation, with cyclophosphamide at 20 mg/kg bw.

An ADG snout-only chamber was used to expose the rats. Each test exposure chamber was housed in a separate extract cabinet to contain any significant release during exposure. A separate chamber was used for each exposure group. Air was introduced into the exposure chamber at 29 liters/minute and extracted from the base at 30 liters/minute. The atmosphere of the test substance was produced by metering the test substance to an all glass vapor generator. The liquid test substance was fed to a fritted glass disc through which dried, filtered air was passed. The vapor generator apparatus was immersed in a water bath maintained at 40 °C. The vapor/air mixture was fed to the exposure chamber. Different exposure concentrations were produced using different liquid feed rates. The target chamber concentrations were achieved by metering the test material from polypropylene syringes mounted on syringe drivers.

The flow of air to each vaporizer was calibrated using precision made tapered glass tube flowmeters and was monitored throughout the exposures using in-line flowmeters. The settings of the test substance metering system required to obtain the target chamber concentrations were determined during the preliminary phase of the study, based on gas-liquid chromatographic (GC) analysis of atmosphere samples. During the 6-hr inhalation exposure, six samples to determine the chamber concentrations of tertiary butyl acetate were collected at approximately hourly intervals. The samples were collected in sequence from each of the three exposure chambers containing the test atmospheres. Chamber temperatures and airflows were monitored continuously and recorded at 30 minute intervals.

The usage of tertiary butyl acetate was recorded, on the day of treatment, for each of the three test groups. The nominal chamber concentration was calculated from these data and the calibrated airflow for each chamber. The chamber concentrations during the study were confirmed by analysis using gas chromatography. The rats were removed from their cages and placed into restraining tubes, which were then attached to the chamber.
Duration of treatment / exposure:
6 hours
Frequency of treatment:
1 day
Post exposure period:
24 and 48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
93 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
451 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
2044 ppm
Basis:
analytical conc.
No. of animals per sex per dose:
10 animals/sex/group for the 1600 ppm test group and negative control; 5 animals/sex/group for the 100 and 400 ppm test groups and the positive control
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control animals were dosed with 20 mg/kg bw of cyclophosphamide by gastric intubation.

Examinations

Tissues and cell types examined:
Following treatment, the animals were examined and any mortalities or clinical signs of reaction were recorded. Bone marrow smears were prepared from 5 males and 5 females in the negative control, each of the test substance groups, and the positive control group sacrificed 24 hours after treatment. In addition, bone marrow smears were prepared from 5 males and 5 females each in the negative control and highest exposure group sacrificed 48 hours after treatment.

Stained smears were examined (under code) for the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. Usually only one smear per animal was examined. The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated mature erythrocytes was also kept.
Details of tissue and slide preparation:
The animals were killed by cervical dislocation following carbon dioxide inhalation and both femurs dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 10 mL Hanks’ balanced salts solution by aspiration through a 21 g needle fitted to a plastic syringe. The resulting cell suspensions were spun at 1000 rpm (150 X g) for 5 minutes. Each resulting cell pellet was resuspended in 2 mL of filtered fetal calf serum before being sedimented out. The final cell pellet was resuspended in a small volume of fetal calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 1976). Several smears were prepared from each femur.

Due to the presence of mast cell granules in rat bone smears, which appear identical to micronuclei when stained using the Romanowsky method, a modified Feulgen staining method was employed for this assay. This method specifically stains DNA-containing bodies deep purple while leaving mast cell granules unstained. This method also allows for reasonable differentiation of mature and immature erythrocytes and produces permanent preparations.

Reference:
Schmid W, 1976. The micronucleus test for cytogenetic analysis. In: Hollander A (ed.) “Chemical Mutagens: Principles and Methods for their Detection”, Vol. 4, p. 31. Published by Plenum Press, New York.
Evaluation criteria:
Micronuclei were identified by the following criteria:
- Large enough to discern morphological characteristics
- Should possess a generally rounded shape with a clearly defined outline
- Should be deeply stained and similar in color to the nuclei of other cells - not black
- Should lie in the same focal plane as the cell
- Lack internal structure, i.e. they are pyknotic
- There should be no micronucleus-like debris in the area surrounding the cell
Statistics:
The results for each treatment group were compared with the results for the concurrent control group using non-parametric statistics. The two sexes were combined to facilitate interpretation and maximize the power of statistical analysis.

For incidences of micronucleated immature erythrocytes: exact one-sided p-values were calculated by permutation (StatXact, CYTEL Software Corp. Cambridge, MA).

Comparisons of several dose levels were made with the concurrent control using the Linear by Linear Association test for trend in a step-down fashion if significance was detected (Agresti et al. 1990).

For assessment of effects on proportion of immature erythrocytes: equivalent permutation tests based on rank scores were used, i.e., exact versions of Wilcoxon's sum of ranks test and Jonckheere's test for trend.

A positive response is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group (P<0.01); individual and/or group mean values should exceed the laboratory historical control range. A negative result is indicated where individual and group mean incidences of micronucleated immature erythrocytes for the group treated with the test substance are not significantly greater than incidences for the concurrent control group (P>0.01) and where these values fall within the historical control range. An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response. Bone marrow cell toxicity was indicated by a statistically significant dose-related decrease in the proportion of immature erythrocytes (P<0.01). This decrease would normally be evident at the 48 hour sampling time.

Reference:
Agresti et al., 1990. Exact inference for contingency tables with ordered categories. Journal of the American Statistical Association, 85:453.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
Clinical signs and mortalities:
No mortalities were observed for any group in this test.

Abnormal clinical signs that were not observed in the negative or positive control animals included red staining around the eyes (low and high exposure levels), eyes half closed (high exposure level only), and unsteady gait (high exposure level only). All exposed animals appeared clinically normal on the morning following exposure. No adverse clinical signs were observed for the positive control animals.

Micronucleated immature erythrocyte counts:
The test substance did not cause any statistically significant increases in the number of micronucleated immature erythrocytes at either sampling time (p > 0.01).
Cyclophosphamide caused large, highly significant increases in the frequency of micronucleated immature erythrocytes (p < 0.001).

Micronucleated mature erythrocytes:
The test substance did not cause any substantial increases in the incidence of micronucleated mature erythrocytes at either sampling time.

Proportion of immature erythrocytes:
The test substance did not cause any significant decreases in the proportion of immature erythrocytes (p > 0.01).
Cyclophosphamide did not cause any statistically significant decreases in the proportion (p < 0.01).

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Tertiary butyl acetate did not cause any statistically significant increases in the numbers of micronucleated immature erythrocytes or any substantial increases in the frequency of micronucleated mature erythrocytes at any concentration tested. Also, the test substance did not cause any significant decrease in the proportion of immature erythrocytes. Cyclophosphamide, the positive control substance, caused large, highly significant increases in the frequency of micronucleated immature erythrocytes.

It is concluded that tertiary butyl acetate did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by snout only inhalation exposure in this in vivo test.

Based on an absence of genotoxic/mutagenic effects in this study, tertiary butyl acetate is not classifiable for Germ Cell Mutagenicity according to Directive 67/548/EEC, the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) or the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
.
Executive summary:

In an in vivo micronucleus assay, groups of male and female rats were subjected to a single 6-hr nose-only inhalation exposure of tertiary butyl acetate at target concentrations of 100, 400 and 1600 ppm. Groups of rats were also exposed to negative and positive controls. No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decrease in the proportion of immature erythrocytes were observed in rats treated with tertiary butyl acetate and sacrificed 24 or 48 hours later, compared to negative control values. The positive control cyclophosphamide caused the appropriate response. It is concluded that tertiary butyl acetate did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by nose-only inhalation exposure at target concentrations up to 1600 ppm.