3-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:

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

Controls

Controls 1

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

Controls 2

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 results

Test results 1

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

Test results 2

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	33.3^5	30.39*
	196.000	50	1033	576	0.55	11.5	33.3^5	22.16*
	253.000	50	1017	585	0.57	11.7	33.3^5	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.