1-nitropropane

Genetic toxicity: in vivo

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:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No data on GLP, comparable to a guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

Genotoxicity of 1- and 2-nitropropane in the rat

Author:

George E, Burlinson B and Gatehouse D.

Year:

1989

Bibliographic source:

Carcinogenesis 10:2329-2334.

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

comparable to a guideline study

GLP compliance:

not specified

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

1-nitropropane

EC Number:

203-544-9

EC Name:

1-nitropropane

Cas Number:

108-03-2

Molecular formula:

C3H7NO2

IUPAC Name:

1-nitropropane

Details on test material:

The test material was used as supplied by Aldrich. Considering the source, it is likely that the material was of high purity.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Animals: Male Sprague-Dawley rats aged 8-12 weeks were used. They were allowed free access to food and water.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Test conduct:
Bone Marrow tests: Two bone marrow tests were conducted. For the first test, groups of 6 animals recevied 100, 200 or 300 mg/kg test material in a volume of 10 ml/kg body weight by gavage. A total of 12 animals/group were dosed. Doses > 100 mg/kg were given as liquid/liquid suspensions due to insolubility. Two negative control groups of 6 animals/each and one positive control group of 4 animals received water or 5 mg/kg cyclophosphamide, respectively. Six animals/group were euthanized at 24 hours and the other six/group were euthanized at 48 hours (with the exception of the positive control group, which was euthanized at 24 hours.

For the second test, groups of 5 animals received 300 or 400 mg/kg test material by gavage. Negative and positive control groups of 3 animals each received water or 5 mg/kg cyclophosphamide, respectively. All groups were euthanized 24 hours after treatment.

Liver micronucleus test: A liver micronucleus test was performed on an additional groups of 8 control animals and 5 animals treated by gavage with 300 mg/kg test material. The test was performed according to the method developed by Braithwaite and Ashby (Mut. Res. 203:23-32). Three days after dosing, the animals received a single oral dose of 1000 mg/kg 4-acetylaminofluorene. Two days later, hepatocytes were isolated accoding to the procedure of Ashby et al (Mut. Res.156:1-18), with the exception that the perfusion buffers were not gassed with air and no antibiotics were added to the buffers.

Duration of treatment / exposure:

single oral gavage

Frequency of treatment:

once

Post exposure period:

24 and 48 hours (bone marrow test)
72 hours (liver micronucleus test)

Doses / concentrations

Remarks:

Doses / Concentrations:
see below
Basis:

No. of animals per sex per dose:

4-8 rats/group

Control animals:

yes, concurrent vehicle

Positive control(s):

bone marrow test-cyclophosphamide
liver micronucleus test- 4-acetylaminofluorene

Examinations

Tissues and cell types examined:

bone marrow and liver

Details of tissue and slide preparation:

Bone marrow- Bone marrow smears were prepared according to procedures listed in Pascoe and Gatehouse, Mut. Res 164:237-243, 1986, and were stained with hematoxylin-eosin. Slides were coded prior to analysis. Two thousand polychromatic erythrocytes (PCE) per slide were analyzed for the presence of micronuclei, and 500 erythrocytes per slide were scored to determine the proportion of PCE among all erythrocytes. Data were analyzed according to methods published by Amphlett and Delow (Mut. Res. 128:161-164).

Liver-Two methods of slide preparation were used. For the first method, hepatocytes were fixed directly with methanol/acetic acid (3:1) and spun in a cetrifuge (50 g) for 2 minutes. The supernatant was removed and the fixation procedure was repeated twice. Slides of the suspension were prepared, checked under phase contrast (10x magnification) for appropriate cell density and air-dried.
For the second method, hepatocytes were allowed to attach for 3-4 hours on coverslips, rinsed with medium, and fixed in 85:10:5 methanol/acetic acid/formaldehyde (37%) for 30 minutes. After this, the coverslips were rinsed with distilled water, allowed to dry and mounted on microscope slides. All slides prepared by this method were placed in 5 M hydrochloric acid for an hour, rinsed in distilled water, and placed in Schiff's reagant for 20 minutes, followed by distilled water for 10 minutes. The cytoplasm was stained with 0.5% light green for 2 minutes, after which the slides were air dried and coded.

For each slide preparation method, a minimum of 2000 hepatocytes per animals were analyzed under 1000x magnification for the presence of micronuclei according to the scoring criteria of Braithwaite and Ashby (Mut. Res. 203:23-32). For the slides prepared by the first method, the number of mitoses was determined in at least 2000 hepatocytes per animal. Data were analyzed for normal distribution and compared using either the Student's t-test or analysis of variance.

Evaluation criteria:

No data

Statistics:

Bone marrow test- Data were analyzed according to methods published by Amphlett and Delow (Mut. Res. 128:161-164).

Liver micronucleous test- Data were analyzed for normal distribution and compared using either the Student's t-test or analysis of variance.

Results and discussion

Test results

Sex:

male

Genotoxicity:

other: negative in the bone marrow test, positive in the liver test (due to increased cell proliferation)

Toxicity:

yes

Remarks:

lethality at 500 mg/kg

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Bone Marrow Tests:
First test: After treatment with the test material, a slight reduction of the percentage of polychromatic erythrocytes was noted for both sampling times in the first study, indicating a cytotoxic effect. There also was a small, dose-related increase in the frequency of micronucleated cells with respect to controls. The 24-hour value for animals treated with 300 mg/kg (1.58 micronucleated PCE/1000 PCE) was significantly different from the vehicle control (0.83 micronucleated PCE/1000 PCE). For the 48 hour harvest, the value for rats treated with 300 mg/kg (1.83 micronucleated PCE/1000 PCE) was slightly (but not significantly) higher than control (0.92 micronucleated PCE/1000 PCE). For both experiments, the frequencies of micronucleated cells in treated animals were within the range of historical controls. The results were valid, since the frequency of micronucleated cells in the positive control at 24 hours (8.4 micronucleated PCE/1000 PCE) was greater than the vehicle control.

Since no signs of general toxicity were observed after treatment with 300 mg/kg, the study was repeated using 400 mg/kg as the highest dose. Whether this dose produced signs of toxicity in the animals was not noted. In the repeat experiment, neither cytotoxicity nor an increase in the frequency of micronucleated cells was noted in animals treated with the test material. Values listed for rats treated with 300 or 400 mg/kg test material were 1.70 and 1.50 micronucleated PCE/1000 PCE, respectively. The frequency of micronucleated cells in the positive control at 24 hours (8.33 micronucleated PCE/1000 PCE) was greater than the vehicle control (1.33 micronucleated PCE/1000 PCE).

Based on the results of the two tests, the authors concluded that the test material did not induce micronuclei in the bone marrow.

Liver Cell Test: Slides from both control and treated animals that were prepared according to the first method had higher frequencies of micronuclei than those prepared by the second method. However, the general outcome of both methods was similar for individual animals and groups. Results of both methods showed a significant increase in the frequency of micronuclei in hepatocytes isolated from rats treated with the test material (17.05 micronucleated cells/1000 hepatocytes in treated animals vs. 7.34 micronucleated cells/1000 hepatocytes in controls for the first method and 14.20 micronucleated cells/1000 hepatocytes in treated animals vs. 5.03 micronucleated cells/1000 hepatocytes in controls for the second method). The elevated micronucleus frequencies in treated animals analyzed by the first method were accompanied by an increased mitotic index (28.85 mitoses/1000 hepatocytes in treated animals vs. 14.92 mitoses/1000 hepatocytes in controls). The frequencies of micronuclei were higher in animals with high mitotic indices.

Any other information on results incl. tables

The mitotic index could not be determined for cells fixed according to the second method since mitotic cells were not retained on the slides.

Since 1-nitropropane is not carcinogenic, the authors suggested that the positive result in hepatocytes was due to increased cell proliferation.  This hypothesis is supported by the increased mitotic index.  The hepatocarcinogen 2-nitropropane also tested negative in the bone marrow test and positive in the hepatocyte test.  However, unlike 1-nitropropane, treatment with 2-nitropropane either had no effect on or inhibited the mitotic index (depending on dose).

This study confirms the negative result for both 1-nitropropane and 2-nitropropane in a mouse bone marrow micronucleus test conducted by Kliesch and Adler (Mut. res 192:181-184, 1987).

The results of Unscheduled DNA Synthesis tests also were discussed in this paper.  1-nitropropane tested negative in this test and 2-nitropropane tested positive.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: negative in bone marrow test and positive in liver test
1-Nitropropane did not induce micronuclei in the bone marrow test. In the liver test, 1-nitropropane resulted in an increase in the frequency of micronuclei in hepatocytes which may be due to increased cell proliferation.

Executive summary:

None