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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline with acceptable restrictions (no data onsubstance purity, no data on cytotoxicity)

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Unnamed
Year:
1987
Reference Type:
review article or handbook
Title:
Unnamed
Year:
1992

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
Deviations:
yes
Remarks:
(no data on cytotoxicity)
Principles of method if other than guideline:
Testing was performed as reported by Loveday et al. (1989) Environ. Molec. Mutagen. 13, 60-94
GLP compliance:
yes
Type of assay:
sister chromatid exchange assay in mammalian cells

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:
3-nitrotoluene
Details on test material:
- Name of test material (as cited in study report): m-nitrotoluene
- Analytical purity: no data

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
(CHO-W-B)
Metabolic activation:
with and without
Metabolic activation system:
S9 was from the livers of Aroclor 1254-induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
Without S9 first trial: 3.8; 11.5; 38.4; 115 µg/ml
Without S9 second trial: 150; 196; 253 µg/ml
With S9 first trial: 38.4; 115, 384 µg/ml

With S9: 354.83; 380.95; 423.28 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s): DMSO
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: With S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: Without S9
Details on test system and experimental conditions:
Each test point was performed in duplicates
In the absence of S9, cells were incubated with o-nitrotoluene or solvent for 2 hours at 37° C. Then 5-Bromodeoxyuridine BrdU was added and incubation was continued for another 23.5 hours making a total incubation time of 25.5h. Cells were washed, fresh medium containing BrdU and Colcemid was added, and incubation was continued for 2 to 3 hours.
In the presence of S9, cells were incubated with o-nitrotoluene or solvent for 2 hours at 37° C. The cells were then washed, and medium containing BrdU was added. Cells were incubated for a further 25.5 hours, with Colcemid present for the final 2 to 3 hours.
Immediately before the cells were harvested, the degree of confluence and availability of mitotic cells were noted. Cells were collected by mitotic shake-off at doses up to the maximum considered likely to yield sufficient metaphase cells for analysis; supernatant medium was returned to appropriate flasks so that subsequent harvests could be made from the same cultures if necessary. Because all mitotic cells were removed in the initial harvest, cells collected during subsequent harvests had come into mitosis during the period between harvests and thus had been exposed to colcemid for an average of 4 hr. After 1-3 min treatment with hypotonic solution (75 mM KCI), cells were fixed in 3:1 methanol : glacial acetic acid (V/V). For a preliminary assessment of cell cycle delay, test slides were prepared from cells treated at the highest dose levels to see if later harvests were necessary. These test slides were stained with "dilute" Hoeschst 33258 (0.5 µg/ml in Sorensen's buffer, pH 6.8) and examined by fluorescence microscopy to assess ceII cycle kinetics. In control cultures, almost all cells completed two cycles in BrdUrd (M2 cells) in 25- 26 hr, whereas, in treated cultures, cell cycle delay was common. In cases of severe delay, additional harvests were made from the same cultures at a later time to obtain sufficient second metaphase (M2) cells for SCE analysis (34 h)
Evaluation criteria:
For individual doses, absolute increases in SCEs per chromosome of 20% or more over the solvent control were considered significant.

Results and discussion

Test resultsopen allclose all
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
in the first and second trial
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Remarks:
positive [Significant increase of SCE rate (approx. 22-31% increase of SCE/chromosome over controls in the presence of metabolic activation)]
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: Chinese hamster Ovary (CHO)
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table1 Induction of Sister Chromatid Exchanges in Chinese Hamster Ovary cells by m-nitrotoluene
 Compound  Dose (µg/mL)  Total cells  No. of Chromosomes  No. of SCEs  SCEs/chromosome  SCEs/cell  Hrs in BrdU Relative SCEs/chromosome (%)² 
 -S9                
 Trial 1                        
 Summary: weak positive             
 Dimethylsulfoxide    50  1031  434  0.42  8.7  25.5  
 Mytomycin-C  0.005  25  511  851  1.66  34.0  25.5  295.62
 m-nitrotoluene  3.840  50  1020  488  0.47  9.8  25.5 13.65
   11.500  50  1029  442  0.42  8.8  25.5  2.04
   38.400  50  986  475  0.48  9.5  25.5  14.44
   115.000  28  556  297  0.53  10.6  25.5  26.90*
 Trial 2                p=0.003*
 Summary: positive                        
 Dimethylsulfoxide    50  1045  477  0.45  9.5  25.5  
 Myromycin-C  0.005  25  519 866   1.66  34.6  25.5  265.56
 m-nitrotoluene  150.000  50  1035  616  0.59  12.3  33.35  30.39*
   196.000  50  1033  576  0.55  11.5  33.35  22.16*
   253.000  50  1017  585  0.57  11.7  33.35  26.02*
 +S9                p=0.001
 Trial 1                
 Summary: negative                        
 Dymethylsulfoxide    50  1003  343  0.34  6.9  25.5  
 Cyclophosphamide  1.5  25  505  522  1.03  20.9  25.5  202.27
 m-nitrotoluene  38.4  50  1021  307  0.30  6.1  25.5  -12.08
   115.0  50  1009  356  0.35  7.1  25.5  3.17
   384.0    989  313  0.31  6.3  25.5  -7.45
                 p=0.625

² SCSs/chromosome of culture exposed to m-nitrotoluene relative to those of culture exposed to solvent

4 Significance of relative SCEs/chromosome tested by regression vs log of the dose.

5 Because of chemical-induced delay in the cell division cycle, harvest time was extended to maximize the proportion of second division cella available for analysis.

* Positive (=20% increase over solvent control)

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
positive without metabolic activation
negative with metabolic activation
Executive summary:

NTP, 1992

3-Nitrotoluene was tested for its capacity to induce SCE in CHO cells with a method similar to current guidelines

3-Nitrotoluene was significant positive in the first trial in the absence of metabolic activation (p=0.003), at the highest tested concentration (115 µg/ml). In the second trail in the absence of metabolic activation (p=0.001), the result was negative in all tested concentration. In the presence of metabolic activation, the result was negative.

Interpretation of results: m-nitrotoluene causes DNA damage in vitro.