Nitrotoluene

Genetic toxicity: in vitro

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

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1987

Reference 2

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

Test material information

Constituent 1

Reference substance name:

2-nitrotoluene

EC Number:

201-853-3

EC Name:

2-nitrotoluene

Cas Number:

88-72-2

Molecular formula:

C7H7NO2

IUPAC Name:

1-methyl-2-nitrobenzene

Test material form:

liquid

Specific details on test material used for the study:

- Name of test material (as cited in study report): o-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: 117; 176; 218; 282 µg/ml
With S9: 354.83; 380.95; 423.28 µg/ml

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

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 results

Test results 1

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

Slight increase of SCE rate (approx 16-18% increase of SCEs/chromosome over controls in the absence of metabolic activation)

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

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

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Induction of Sister Chromatid Exchanges In Chinese Hamster Ovary Cells by o-Nitrotoluene (Without S9 activation)

Without S9
Compound	Dose (µg/ml)	Total cells	No of SCEs	SCEs/ chromosome	SCEs/ cell	Hrs in BrdU	Relative SCEs/ chromosome
DMSO	-	50	485	0.46	9.7	25.5
Mitomycin C	0.005	50	2236	2.15	44.7	25.5	360.15
o-nitrotoluene	117	50	569	0.54	11.4	34	16.87
	176	50	557	0.54	11.1	34	16.42
	218	50	576	0.55	11.5	34	18.76
	282	0

Table 2: Induction of Sister Chromatid Exchanges In Chinese Hamster Ovary Cells by o-Nitrotoluene (With S9 activation)

With S9
Conpound	Dose (µg/ml)	Total cells	No of SCEs	SCEs/ chromosome	SCEs/ cell	Hrs in BrdU	Relative SCEs/ chromosome
DMSO	-	50	380	0.36	7.6	25.5
cyclophosphamide	1.50	50	1651	1.57	33.0	25.5	334.89*
o-nitrotoluene	354.83	50	499	0.47	10.0	25.5	31.32*
	380.95	50	467	0.44	9.3	25.5	22.90*
	423.28	50	488	0.46	9.8	25.5	28.79*

* > = 20% increase in SCEs/chromosome

Applicant's summary and conclusion

Executive summary:

SCE was run on Chinese Hamster Ovary cells (CHO) with a method similar to OECD guideline 479 with acceptable restrictions (no data on cytotoxicity). 2-Nitrotoluene was positive in the presence of metabolic activation with >= 20% increase in SCEs, at all tested concentration (354.83 - 423.28 µg/ml). In the absence of metabolic activation, the result was negative (approx.16 - 18% increase of SCE). O-nitrotoluene causes DNA damage in vitro.