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Toxicological information

Genetic toxicity: in vivo

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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: Not GLP, comparable to a guideline study.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
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:
no
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 and environmental conditions:
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:
Bone Marrow Tests: Groups of 5-6 animals received 50 or 100 mg/kg test material in a volume of 10 ml/kg body weight by gavage. One group of 11 animals was given 300 mg/kg test material. 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 8 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). There was no 300 mg/kg group for the 48 hour test point.

Liver Micronucleus Test: A liver micronucleus test was performed on an additional group of 8 control animals and groups of 6-7 animals treated by gavage with 25, 50 or 75 mg/kg test material. The test was performed according to the method developed by Braithwaite and Ashby (Mut. Res. 203:23-32, 1988). Three days after dosing, the animals received a single oral dose of 1000 mg/kg 4-acetylaminofluorene. Two days later, hepatocytes were isolated according to the procedure of Ashby et al (Mut. Res.156:1-18, 1985), with the exception that the perfusion buffers were not gassed with air and did not contain antibiotics. No positive control was noted.
Duration of treatment / exposure:
up to 48 hours (bone marrow test) and 72 hours (liver micronucleus test)
Frequency of treatment:
see "details on exposure"
Post exposure period:
None
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 50, 100 or 300 mg/kg (bone marrow micronucleus)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 25, 50 or 75 mg/kg (liver micronucleus)
Basis:
nominal conc.
No. of animals per sex per dose:
Bone Marrow Micronucleus Test: 5-6 rats/50 or 100 mg/kg, 11 rats/300 mg/kg, 6 rats/negative control groups and 4 rats/positive control

Liver Micronucleus Test: 6-7 rats/dose group and 8 rats/control
Control animals:
yes
Positive control(s):
Bone Marrow Micronucleus Test: cyclophosphamide
Liver Micronucleus Test: no data

Examinations

Tissues and cell types examined:
liver and bone marrow cells
Details of tissue and slide preparation:
Bone Marrow Micronucleus Test: 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.

Liver Micronucleus Test: 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 centrifuge (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 reagent 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, 1988). For the slides prepared by the first method, the number of mitoses was determined in at least 2000 hepatocytes per animal.
Evaluation criteria:
Bone Marrow Micronucleus Test: Data were analyzed according to methods published by Amphlett and Delow (Mut. Res. 128:161-164, 1984).

Liver Micronucleus Test: 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, 1988). For the slides prepared by the first method, the number of mitoses was determined in at least 2000 hepatocytes per animal.
Statistics:
Liver Micronucleus Test: Data were analyzed for normal distribution and compared using either the Student's t-test or analysis of variance. The critical level of significance was p < 0.05.

Results and discussion

Test results
Sex:
male
Genotoxicity:
other: Ambiguous- Negative in bone marrow test and positive in liver micronucleus test
Toxicity:
yes
Remarks:
cytotoxic effect at 300 mg/kg
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
Bone Marrow Test: Only 5 animals in the 300 mg/kg group survived for 24 hours. Therefore, there were no results for the 48 hour time period for this group. IN the 300 mg/kg group, there was a slight increase in the frequency of micronuclei (2.10 +/- 1.14 micronucleated PCE/1000 PCE in treated vs. 1.08 +/- 1.02 micronucleated PCE/1000 PCE in control). However, the average value for the treated animals was not significantly different from the study control and data from only one animal was outside of the historical control range. The results for the other groups were negative. There was no significant effect of treatment on the percentages of PCE. However, in the bone marrow smears of the 300 mg/kg group, an increased incidence of pycnotic erythroblasts was observed, indicative of a cytotoxic effect. The positive control (CP) induced a significant increase in micronuclei frequency, validating the test system and method.

Based on the results of the test, 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 the first method showed a significant increase in the frequency of micronuclei in hepatocytes isolated from rats treated with 25 mg/kg (14.25 +/- 5.46 micronucleated cells/1000 cells in treated vs. 7.34 +/- 1.75 micronucleated cells/1000 cells in control) or 50 mg/kg (32.33 +/- 6.61 micronucleated cells/1000 cells in treated vs. 7.34 +/- 1.75 micronucleated cells/1000 cells in control) test material. The value at 75 mg/kg (24.86 +/- 10.13) also was higher than control, but not significantly. The mitotic index of cells from rats exposed to 25 mg/kg (8.83 +/- 1.43) and 75 mg/kg (9.29 +/- 2.20) was significantly lower than control (14.92 +/- 1.39).

For the second method, only the results at 50 mg/kg (21.83 +/- 4.58 micronucleated cells/1000 cells) were significantly different from control (5.03 +/- 0.90 micronucleated cells/1000 cells). At 25 and 75 mg/kg, the numbers of micronucleated cells were increased in 2/6 and 4/7 animals, respectively. The average values in these group were elevated (8.08 +/- 2.76 and 14.53 +/- 4.54 micronucleated cells/1000 cells, respectively), but were not significantly different from the control value listed above.

Based on the results of the test, the authors concluded that the test material induced micronuclei in hepatocytes.

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.

1-Nitropropane also tested negative in the bone marrow and positive in the liver.
  However, unlike 2-nitropropane, 1-nitropropane produced an increase in the mitotic index of the liver. 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): ambiguous
Negative in bone marrow micronucleus test and positive in liver micronucleus test.
Executive summary:

None