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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:
circa 1997
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to guideline and/or standard method but was non-GLP.

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Toxicology and carcinogenesis studies of nitromethane (CAS No. 75-52-5) in F344/N rats and B6C3F1 mice.
Author:
National Toxicology Program
Year:
1997
Bibliographic source:
National Toxicology Program Technical Report Series No. 461. U.S. Department of Health and Human Services (USDHHS), Public Health Service, National Institute of Health (NIH), NIH Publication No. 97-3377, dated February 1997.
Reference Type:
publication
Title:
Micronucleated erythrocyte frequency in peripheral blood of B6C3F1 mice from short-term, prechronic, and chronic studies of the NTP carcinogenesis bioassay program.
Author:
Witt KL, Knapton A, Wehr CM et al.
Year:
2000
Bibliographic source:
Enivron Molec Mutagen 36:163-194

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
>98% nitromethane

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
The mice were 4 weeks old on receipt from the supplier. They were quarantined for 13-14 days before use. Five animals per sex were randomly selected for parasite evaluation and gross examination for evidence of disease. The kidneys of 5 animals/sex were screened to ensure genetic integrity. At the end of the study, serologic analyses were performed on 5 control mice/sex. Water and food were available ad libitum (except during exposure, when food was withheld).

Administration / exposure

Route of administration:
inhalation: vapour
Vehicle:
None
Details on exposure:
The test material was held in a stainless-steel reservoir under a nitrogen blanket. The material was pumped through a liquid distribution manifold of stainless steel tubing to heated-wick vaporizers.One set of dual vaporizers supplied vapor to all chambers. The vapor-laden air was transferred through the distribution line and diluted with HEPA- and charcoal-filtered air. Three-way valves in the chamber inlet ducts allowed nitromethane vapors to be diverted to the exhaust until a stable concentration of test material was built up in the distribution line. At each chamber, vapor moving through the inlet duct was further diluted with filtered air to the appropriate concentration of test material with a metered three-way valve. A small particle detector was placed in the chambers to measure concentrations of aerosol. No particle counts above the minimum resolvable level (200 particles/cm3) were detected.
Duration of treatment / exposure:
6 hours/day
Frequency of treatment:
5 days/week for 13 weeks
Post exposure period:
None
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 94, 188, 375, 750, or 1,500 ppm nitromethane
Basis:
nominal conc.
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Positive control(s):
URNE was tested as the positive control with nitromethane according to the NTP web site. The web site does not identify URNE.

Examinations

Tissues and cell types examined:
Peripheral blood samples were obtained from male and female B6C3F, mice at the end of the 13-week toxicity study. Smears were immediately prepared and fixed in absolute methanol and were later stained with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y (MacGregor et al., 1983).
A detailed discussion of this assay can be found in MacGregor et al. (1990).
Details of tissue and slide preparation:
Peripheral blood samples were obtained from male and female B6C3F1 mice at the end of the 13 -week toxicity study. Smears were immediately prepared and fixed in absolute methanol and were later stained with a chromatin-specific fluorescent dye mixture of Hoechst 33258/pyronin Y (MacGregor et al., 1983) and coded. Slides were scanned to determine the frequency of micronuclei in 2,000 normochromatic erythrocytes (NCEs) in each animal per dose group. The criteria of Schmid (1976) were used to define micronuclei, with the additional requirement that the micronuclei exhibit the characteristic fluorescent emissions of DNA (blue
with 360 nm and orange with 510 nm ultraviolet illumination); the minimum size limit was approximately one-twentieth the diameter of the NCE cell.
Evaluation criteria:
In the micronucleus test, an individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any exposure group is less than or equal to 0.025 divided by the number of exposed groups. A final call of positive for micronucleus induction is preferably based on reproducibly positive trials (as noted above). Ultimately, the final call is determined by the scientific staff after considering the results of statisti.ca1 analyses, the reproducibility of any effects observed, and the magnitudes of those effects.
Statistics:
The results were tabulated as the mean of the pooled results from all animals within a treatment group, plus or minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a statistical software package that tested for increasing trend over exposure groups with a one tailed Cochran-Armitage trend test, followed by pairwise comparisons between each exposure group and the control group (Margolin et al., 1990). In the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the Cochran-Armitage test was adjusted upward in proportion to the excess variation.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
not specified
Additional information on results:
Nitromethane administered by inhalation for 13 weeks at concentrations up to 1,500 ppm did not induce increased frequencies of micronucleated erythrocytes in the peripheral blood of male or female mice.

Any other information on results incl. tables

Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Treatment with Nitromethane by Inhalation for 13 Weeks

   

 Micronucleated NCEs/Total NCEs (%)   

 
    % PCE
 Dose (ppm)  Male  Female  Male  Female
 0  0.052 + 0.0076  0.055 + 0.0071  1.04 + 0.09  1.42 + 0.07
 94  0.080 + 0.0078p = 0.006  0.037 + 0.0062p = 0.974  1.25 + 0.09  1.24 + 0.09
 188  0.061 + 0.0064p = 0.198  0.040 + 0.0068p = 0.934  1.21 + 0.06  1.27 + 0.11
 375  0.067 + 0.0111p= 0.075  0.039 + 0.0031p = 0.949  1.21 + 0.05  1.43 + 0.08
 750  0.064 + 0.0076p = 0.125  0.055 + 0.0056p = 0.496  1.30 + 0.04  1.60 + 0.09
 1500  0.070 + 0.0066p = 0.049  0.049 + 0.0064p = 0.711  1.46 + 0.12  1.85 + 0.08
 Trend test  P = 0.273*  P = 0.186    
 ANOVA    P = 0.021    P < 0.001
 Positive control (URNE according to NTP website)  3.27 + 0.53p <0.0001      

 * Significance of micronucleated NCEs/total NCEs tested by a one-tailed trend test; significant at P<0.025

NTP website is http://ntp-apps.niehs.nih.gov/ntp_tox/index.cfm?fuseaction=micronucleus.micronucleusData&cas_no=75%2D52%2D5&endpointlist=MN

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Nitromethane administered by inhalation for 13 weeks at concentrations up to 1,500 ppm did not induce increased frequencies of micronucleated erythrocytes in the peripheral blood of male or female mice.
Executive summary:

The genotoxic potential of nitroethane in the mouse erythrocyte micronucleus assay was evaluated. Nitromethane administered by inhalation for 13 weeks at concentrations up to 1,500 ppm did not induce increased frequencies of micronucleated erythrocytes in the peripheral blood of male or female mice.