Heptan-2-one

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26/3/1996 to 13/5/1996 for the analytical phase

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to acceptable scientific standards and GLPs.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

This study was conducted to evaluate the ability of 2-heptanone to induce covalent DNA adducts in the livers of female Fischer 344 rats exposed to the test material by inhalation. Nuclear DNA was isolated from the livers of control and 2-heptanone exposed (1000 ppm) female Fischer 344 rats (5 females/group), and analyzed for the presence of DNA adducts by the ^32P-postlabeling assay with and without enrichment of adducts in the DNA digests. In the non-enrichment method, total nucleotides (normal and adducted) were labeled and resolved by TLC. In the enrichment method, an anion-exchange chromatographic procedure was utilized to isolate N7-alkylpurine nucleotide adducts from the DNA digests prior to ^32P-labeling and TLC.

GLP compliance:

yes

Type of assay:

other: DNA adduct

Test material

Test material information

Constituent 1

Reference substance name:

Heptan-2-one

EC Number:

203-767-1

EC Name:

Heptan-2-one

Cas Number:

110-43-0

Molecular formula:

C7H14O

IUPAC Name:

heptan-2-one

Constituent 2

Reference substance name:

606-024-00-3

IUPAC Name:

606-024-00-3

Constituent 3

Reference substance name:

methyl amyl ketone; methyl pentyl ketone; MAK; 1-methylhexanal; butylacetone

IUPAC Name:

methyl amyl ketone; methyl pentyl ketone; MAK; 1-methylhexanal; butylacetone

Details on test material:

- Name of test material (as cited in study report): 2-heptanone

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

female

Details on test animals or test system and environmental conditions:

No information was provided in study report.

Administration / exposure

Route of administration:

inhalation: vapour

Vehicle:

Air

Details on exposure:

Groups of five female Fischer 344 rats were exposed to air only (negative control) or one of three concentrations of 2-heptanone, 6 hours/day, 5 days/week for 2 weeks.

Duration of treatment / exposure:

6 hours

Frequency of treatment:

5 days/week for two weeks

Post exposure period:

Livers were harvested at study termination and liver nuclear pellets prepared and stored at -70 ºC until use. Pellets from rats exposed to 1000 ppm (the highest concentration) were analyzed first for the presence of DNA adducts. Pellets from animals exposed to the lower two concentrations (80 and 400 ppm) of 2-heptanone were only analyzed if results at the highest concentration were positive.

Doses / concentrations

Remarks:

Doses / Concentrations:
80, 400 and 1000 ppm
Basis:
no data

No. of animals per sex per dose:

5 females/exposure group

Control animals:

other: For inhalation exposure, negative control animals were exposed to air only. For the analytical method, commercially available calf thymus DNA served as the negative control.

Positive control(s):

The positive control article for the analytical method was calf thymus DNA modified in vitro with 2-heptylene oxide.

Examinations

Tissues and cell types examined:

Liver nuclei were analyzed for the presence of DNA adducts.

Details of tissue and slide preparation:

Positive Control:
The positive control article was calf thymus DNA modified in vitro with 2-heptylene oxide, prepared as reported by Kumar et al. (1995). It was obtained from the Karolinska Institute, Stockholm, Sweden. The identity of this control was established by the detection of an adduct(s) not present in the DNA prior to reaction with 2-heptylene oxide and by comparison of the ^32P-postlabeling pattern with published results for this modified DNA (Kumar et al., 1995; Eide et al., 1995). Commercially available calf thymus DNA was analyzed in triplicate as a negative control for the analytical method. The level of modification of the positive control material received was 43 adducts per 10^6 DNA nucleotides. The positive control DNA was stored for approximately the same length of time as the rat liver nuclear fractions and this served as indirect evidence for the stability of any adducts that may have been present in the liver nuclear pellets.

References:
Kumar et al., 1995. ^32P-Postlabeling method for the detection of 7-alkylguanine adducts formed by the reaction of different 1,2-alkyl epoxides with DNA. Carcinogenesis, 16:483-489.
Eide et al.,1995. Uptake, distribution, and formation of hemoglobin and DNA adducts after inhalation of C2-C8 1-alkenes (olefins) in the rat. Carcinogenesis, 6:1603-1609.

Isolation of DNA:
Liver DNA was obtained from the liver nuclear pellets of the five female Fischer 344 rats exposed to the highest concentration of 2-heptanone using isolation methods described by Reddy and Randerath (1994). The liver pellets were suspended in sodium dodecyl sulfate, EDTA and Tris-HCl and homogenized for 15-30 seconds. Ribonuclease A and ribonuclease T1 were added to each homogenate and the samples were incubated at 36 ºC for 40 minutes. Following addition of proteinase K, samples were incubated for an additional 40 minutes and then extracted with phenol and Sevag. DNA was precipitated using NaCl and absolute ethanol. The DNA pellet was washed with ethanol and dissolved in water. The concentration of the purified DNA was characterized by UV absorbance measurements.

References:
Reddy MV and Randerath K, 1987. ^32P-Analysis of DNA adducts in somatic and reproductive tissues of rats treated with the anticancer antibiotic, mitomycin C. Mutat. Res., 179:75-88.

DNA adduct detection procedures:
DNA samples were analyzed for adducts with and without pre-purification of DNA digests. In the analysis involving no purification of DNA digests to 3’-mononucleotides, total DNA nucleotides were digested, labeled, and resolved by TLC (Kumar et al., 1995; Szyfter et al., 1994). In the enrichment method involving purification of DNA digests prior to labeling, N7-alkylpurine adducts were isolated by strong anion-exchange column chromatography.

References:
Szyfter et al., 1994. ^32P-postlabeling analysis of DNA adducts in humans: adduct distribution and method development. Mutat. Res., 313:269-276.

^32P-labeling:
For labeling by the non-enrichment method, the DNA digests were diluted to 1 pmole DNA nucleotides/µL, and 2 µL of the diluted DNA digest were dried and subjected to 32^P-labeling. In the case of the positive control, 2 µL of the digest were spiked with a 1µL solution containing 14 fmoles of purified N7-alkylpurine nucleotide adduct to increase the adduct level, and the solution was dried and 32^P-labeled. The purified adduct was prepared by anion-exchange chromatography of 2-heptylene oxide-modified DNA digests. To convert ^32P-labeled 3’,5’-bisphosphate nucleotide adducts to ^32P-labeled 5’-mononucleotide adducts, 3 µL of ^32P-labeled solution were treated with a 1.5 µL solution containing nuclease P1, ammonium formate, ZnCl2, and water. The reaction mixture was incubated at 37 ºC for 15 minutes.

Separation of ^32P-labeled adducts by Thin Layer Chromatography (TLC):
Adducts were resolved by two dimensional polyethyleneimine(PEI)-cellulose TLC. The labeled DNA digests were applied to PEI-cellulose sheets and developed in the first dimension in 0.1M ammonium formate to a 10-cm top. The sheets were dried and developed in the second dimension in 0.5M ammonium formate and n-propanol to a 20-cm top and dried. The radioactivity was visualized by autoradiography. After 2-dimensional separation and evaluation of results, the TLC plates obtained by the non-enrichment method were further developed in 0.5M ammonium formate to a 10-cm top, dried, and then developed perpendicular to the preceding direction in 0.45M lithium chloride to a 20-cm top. The plates were allowed to dry and subjected to autoradiography at room temperature and/or <-70 °C. Room temperature was chosen over <-70 ºC in some instances to improve resolution of spots and to avoid over exposure to X-ray films.

Separation of normal nucleotides:
To ensure that DNA was hydrolyzed and that [γ-^32P]ATP was present in excess during ^32-P-labeling, an aliquot of each diluted labeled DNA digest was applied to PEI-cellulose TLC plates and developed in 0.25M lithium chloride and 0.12M sodium phosphate. The plates were then dried and autoradiographed to locate radioactivity.

Evaluation criteria:

Assay acceptance criteria:
An assay was considered acceptable for evaluation of the test results only if the following criteria were satisfied:
-the positive control yielded a positive response on the thin-layer chromatogram, as shown by the presence of spot(s) not present on the untreated DNA control map. If a spot(s) was common to both treated and control TLC maps, but was intensified in the treated TLC map, a significant increase in radioactivity was considered a positive response.
-the sample DNA was digested, and ATP was present in excess during ^32P-labeling, as evaluated by TLC.

Response evaluation criteria:
A test substance was considered to induce DNA adducts if one or more spots were obtained which were not observed on chromatograms obtained from the negative controls. Significant intensification of background spots after treatment was also considered a positive response. Adducts may be observed using either the enrichment or non-enrichment methods. If no significant differences in chromatograms were observed between a test substance and the negative control, the test substance was evaluated as not containing detectable levels of DNA adducts.

Statistics:

The significance of a difference in adduct levels between control and 2-heptanone treated groups was evaluated by a two-tailed t-test on the ranked data. A probability (P) value of 0.05 or less was considered to represent a significant response.

Results and discussion

Test results

Sex:

female

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Representative autoradiograms of DNA samples from control and 2-heptanone-exposed rats for the non-enrichment and enrichment methods were similar to those obtained with the corresponding control DNA samples and there were no extra spots indicative of DNA adducts in the treated DNA samples. Intensification of background spots was not detected after treatment, indicating the absence of adduct formation. The ^32P-maps obtained by the non-enrichment method were further developed in stronger solvents to allow higher migration and resolution of radioactivity near the origin. Neither an extra spot nor intensification of any spot was seen in the maps of treated DNA samples compared with the maps of control DNA samples, indicating that adduct formation from 2-heptanone exposure did not occur.

Postlabeling experiments were performed in three trials. In Trial 1, adducts were detected with the positive control DNA by the non-enrichment method, but not by the enrichment method. Therefore, the analysis by the enrichment method was repeated in Trials 2 and 3, and the positive control showed adducts in Trial 3 though not in Trial 2.

Using both enrichment (Trial 3) and non-enrichment (Trial 1) methods, extra spots (3 or possibly 4) indicative of adducts were detected with the positive control DNA modified with 2-heptylene oxide, and these spots were not present in the control. Adduct migration was higher in Trial 3 compared with Trial 1, which was probably due to the variation between different batches of TLC plates. Analysis by scintillation counting revealed that the DNA modification levels were significantly lower by the enrichment method than by the non-enrichment method. Separation of labeled DNA digests by one-dimensional TLC in 0.25M LiCl and 0.12M sodium phosphate showed ATP spots, demonstrating that there was excess ATP during labeling. The TLC maps also revealed the presence of normal nucleotides with the non-enrichment method, but few with the enrichment method, indicating that DNA digestion was essentially complete.

Since DNA adducts were not detected with the highest inhalation exposure level used (1000 ppm), analyses of adducts for the two lower exposure levels (80 and 400 ppm) were not pursued.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test material, 2-heptanone (methyl n-amyl ketone), did not produce DNA adducts in the livers of female Fischer 344 rats repeatedly exposed to 2-heptanone via inhalation, as measured in this study using ^32P-postlabeling assay techniques.

2-Heptanone is not classified for genotoxic or mutagenic effects in Annex I of Directive 67/548/EEC. Based on the absence of genotoxic or mutagenic effects in this study, 2-heptanone (methyl n-amyl ketone) is not classified for Germ Cell Mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) 1272/2008 or the UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Executive summary:

This study was conducted to evaluate the ability of 2-heptanone to induce covalent DNA adducts in the livers of female Fischer 344 rats exposed to the test material by inhalation. Nuclear DNA was isolated from the livers of control and 2-heptanone exposed (1000 ppm) female Fischer 344 rats (5 females/group), and analyzed for the presence of DNA adducts by the ^32P-postlabeling assay with and without enrichment of adducts in the DNA digests. In the non-enrichment method, total nucleotides (normal and adducted) were labeled and resolved by TLC. In the enrichment method, an anion-exchange chromatographic procedure was utilized to isolate N7-alkylpurine nucleotide adducts from the DNA digests prior to ^32P-labeling and resolution by TLC. Using both enrichment and non-enrichment methods, extra spots (3 or possibly 4) indicative of adducts were detected with the positive control DNA modified with 2-heptylene oxide, and these spots were not present in the control. No spots indicative of DNA adduct formation were seen in the DNA obtained from livers of 2-heptanone exposed rats. Under the conditions of the study, 2-heptanone did not produce DNA adducts in the livers of female Fischer 344 rats after repeated inhalation exposure to 1000 ppm 2-heptanone.