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Toxicological information

Genetic toxicity: in vivo

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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication which meets basic scientific principles
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
NTP Technical Report on Toxicity Studies of o-, m-, and p-Nitrotoluenes (CAS Nos.: 88-72-2, 99-08-1, 99-99-0). Administered in Dosed Feed to F344/N Rats and B6C3F1 Mice.
Author:
NTP
Year:
1992
Bibliographic source:
NTP Technical Report Tox 23, NIH Publication, No. 93-3346, U.S. Department of Health and Human Sevices

Materials and methods

Principles of method if other than guideline:
Study of the ability of m-nitrotoluene to induce s-phase and unscheduled DNA synthesis in hepatocytes
GLP compliance:
not specified
Type of assay:
unscheduled DNA synthesis

Test material

Constituent 1
Chemical structure
Reference substance name:
3-nitrotoluene
EC Number:
202-728-6
EC Name:
3-nitrotoluene
Cas Number:
99-08-1
Molecular formula:
C7H7NO2
IUPAC Name:
1-methyl-3-nitrobenzene
Details on test material:
- Name of test material (as cited in study report): m-nitrotoluene
- Analytical purity: > 96%

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic Farms, Inc. (Germantown, NY, USA)
- Age at study initiation: 11 to 12 weeks
- Assigned to test groups randomly: [yes, under following basis: computer-generated randomization procedure]
- Housing: 3 per cage
- Acclimation period: 7 to 8 weeks

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used:no data
Details on exposure:
Animals were weighed prior to dosing and at termination, and were observed for toxic effects.
At the time points specified, animals were anesthetized with sodium pentobarbitol, and the livers were perfused with Hanks´ balanced salts containing ethylene glycol-bis(baminoethylether)-N,N-tetra-acetic acid (EGTA) and HEPES buffer at pH 7.2 for 2 - 4 minutes and with a collagenase solution for 4-10 minutes. The liver was removed and hepatocytes were obtained by mechanical dispersion of the excised liver tissue in Williams´ Medium C with collagenase.
Cells were cultured on plastic coverslips in Williams Medium E for 1.5 to 2 hours at 35¡C.
Unattached cells were then removed and cultures refed with 2.5 ml Williams´ Medium E (without fetal bovine serum) containing 3H-thymidine. Attachment efficiency was determined for each culture using trypan blue dye exclusion in situ; all cultures were found to contain >80% viable cells.
After a labeling period of approximately 4 hours, the cultures were refed with Williams´ Medium E (without fetal bovine serum) containing unlabeled thymidine, and returned to the incubator for 14 to 19 hours
Duration of treatment / exposure:
12 hours
Frequency of treatment:
on single exposure
Post exposure period:
no data
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 200, 500 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
3
Control animals:
yes, concurrent no treatment
Positive control(s):
no data

Examinations

Tissues and cell types examined:
The liver was removed and hepatocytes were obtained
Details of tissue and slide preparation:
The nuclei were swollen by the addition of 1% sodium citrate to the dishes containing the coverslips, for 8-12 minutes, after which the cells were fixed in glacial acetic acid:ethanol (1:3), washed with deionized water, and dried for at least 24 hours. The coverslips were mounted on glass slides, dipped in Kodak NTB2 photographic emulsion, and dried. Coated slides were stored for 7 days at 4¡C in light-tight boxes containing Drierite. The emulsions were developed in D19, fixed, and stained with Williams modified hematoxylin and eosin procedure.
Evaluation criteria:
The cells were examined microscopically at approximately 1500X magnification under oil immersion. UDS was measured by counting nuclear grains and subtracting the largest number of grains from 3 nuclear-sized areas adjacent to each nucleus (background count). This value is referred to as the net nuclear grain count (NNG) and can be a negative number if the number of grains in any background area exceeds the number of grains in the nucleus. The NNG was determined for 30 randomly selected cells on each coverslip. The mean NNG was determined from triplicate coverslips, if available (90 total nuclei), for each treated animal (2 or 3 animals per dose level).
The test was considered positive if an increase in the mean net nuclear grain count was observed to at least 5 grains per nucleus in excess of the concurrent vehicle control, or the percentage of nuclei with 5 or more net grains was increased above 10% of the examined population, in excess of the concurrent vehicle control.
The test is considered negative if the mean net grain count for all groups is less than 1 above the concurrent control value, and/or the percent of nuclei with 5 or more net grain counts does not increase more than 2% above the concurrent vehicle control.
The percentage of cells in S-phase was calculated as those cells exhibiting nuclei blackened by grains too numerous to count. Approximately 2000 cells were counted from randomly selected areas of each slide. For each dose, 3 slides were scored for each of 3 animals (6000 total cells).
Statistics:
Significance of the response was determined using the Student´s t-test modified for unpaired observations with unequal variances.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
No induction of UDS were seen in the hepatocytes.

Any other information on results incl. tables

Unscheduled DNA Synthesis in male rats 12 hours after single oral gavage dose of m-nitrotoluene

 Dose (mg/kg)  m-nitrotoluenea
 0 -3.77 ± 0.35
 100  -3.84  ± 0.23
 200  -2.66  ± 0.73
 500  -3.44  ± 0.60

a Figure indicate mean nuclear grain count  ± standard deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Executive summary:

NTP 1992

Groups of 3 male F344 rats recevied a single oral dose of 200 or 500 mg m-nitrotoluene bw by gavage. 12 hours after administration the animals were sacrificed, the hepatocytes isolated and DNA repair synthesis rates measured.

m-nitrotoluene showed no effect at either dose in this experiment