3-nitrotoluene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Reverse gene mutation assay; OECD 471: Negative (Key: Haworth, 1983, rel.2)

Sister Chromatid Exchange Assay: Negative (Key: NTP, 1992, rel. 2)

Link to relevant study records

Reference

Reference 1

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)

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

Target gene:

not applicable

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

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.

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'.

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)

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guidelien study with acceptable deviations

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only 4 strain tested instead of 5. Positive control: 2-aminoantracene was only indicator of efficiency of S9-mix. Individual plate reading not reported.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

his

Species / strain / cell type:

other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

microsomal enzyme reaction mix (S9-mix)

Test concentrations with justification for top dose:

EGG (LABORATORY): 0; 3.3; 10.0; 33.0; 100.0; 333.0 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA): in all strains in presence of rat and hamster S-9. 4-Nitro-o-phenylenediamine (NOPD): on TA98, without S-9. sodium azide (SA) : on TA100 and TA 1535, without S-9. 9-aminoacridine (9-AAD) was tested on 1537, without S-9.

Remarks:

The actual concentration for each positive control chemical used for each strain and activation condition was selected by the individual laboratory based on dose-response curves generated at the beginning of the testing program (see table 1)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48h
- Selection time (if incubation with a selection agent): 48h
- Concentration of S9 mix: 10% for both Hamster S9 mix and Rat S9 mix

SELECTION AGENT (mutation assays): L- histidine

NUMBER OF REPLICATIONS: 2 trial per strain and 3 dishes per dose


DETERMINATION OF CYTOTOXICITY
- Method: other: viability on complete medium and reduced numbers of revertant colonies per plate and/or thinning or absence of the bacterial lawn

Evaluation criteria:

A positive response was indicated by a reproducible, dose-related increase, whether it be twofold over background or not.
An equivocal response was defined as an increase in revertants which was not dose-related, not reproducible, or was of insufficient magnitude to support a determination of mutagenicity. A negative response was obtained when no increase in revertant colonies is observed following chemical treatment.

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: to select the dose range for the mutagenesis assay, the test chemicals were checked for toxicity to TA 100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S-9 mix. If toxicity was not apparent in the preliminary toxicity determination, the highest dose tested was 10 mg/plate; otherwise the upper limit of solubility was used. If toxicity was observed, the doses of test chemical were chosen so that the high dose exhibited some degree of toxicity. Occasionally, in the earlier tests, the high dose was greater than 10 mg/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537

TA 100

TA 1535

TA 1537

TA 98

Dose

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

0.0

142±4.6

105±8.5

106±5.9

29±4.4

10±3.2

13±1.5

5±0.9

6±1.5

9±2.3

21±3.6

25±3.5

29±2.8

3.3

135±6.0

126±4.5

110±5.7

35±2.2

10±2.0

12±0.9

8±0.3

6±0.3

11±1.8

15±0.3

29±0.9

25±1.5

10.0

136±2.5

115±9.6

110±3.8

29±6.0

11±2.1

11±1.3

5±1.2

7±2.4

10±1.9

20±3.1

24±1.9

26±1.5

33.3

151±6.1

128±3.7

105±7.7

33±1.2

9±0.7

8±0.9

7±1.2

7±1.2

6±1.7

19±1.8

32±2.2

25±3.4

100.0

143±10.4

125±1.3

116±5.0

30±1.5

11±0.6

8±0.3

3±1.0

8±2.3

13±2.5

22±1.8

24±4.6

28±1.2

333.3

120±8.4s

133±12.3s

106±9.5s

26±0.7s

10±1.2

13±1.7

2±0.9s

6±2.3

9±0.3

13±1.7s

23±4.5

27±3.3s

POS

2080±58.4

994±38.4

2190±78.4

1426±28.3

58±2.2

167±9.0

469±107.7

60±6.2

262±12.5

1707±61.0

779±32.7

2059±84.9

 

Table 1.Mutagenic responses of Salmonella strains TA100, TA 1535, TA 1537, and TA 98 (mean ± SEM) to m-nitrotoluene.

 Abbreviations: NA, not activated; RLI, rat liver S-9, Aroclor1254 induced; HLI, hamster liver S-9, Aroclor1254 induced, t=complete clearing background

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Executive summary:

Haworth (1983)

 

3-nitrotoluene was tested in the Ames test similar to OECD guideline 471 with deviations (Only 4 bacterial strains were used. The highest dose tested was 10 mg/plate. 2-Aminoanthracene was tested as sole indicator of the efficacy of the S9-mix. 4-Nitro-o-phenylenediamine was tested on TA 98), using preincubation method, in strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimirium.

Liver S9 fractions were prepared from male Sprague-Dawley rats and male Syrian hamsters that were injected, i.p., with Aroclor 1254. The concentrations of 3-nitrotoluene used were 0.0, 3.3, 10.0, 33.0, 100.0 and 333.0 µg/plate. The results were negative, 333.0 µg/plate resulted toxic for the strains TA 100 (with and without S9 mix), TA 1535 (without S9 mix), TA 1537 (without S9 mix) and TA 98[with (hamster) and without S9 mix].

Genetic toxicity in vivo

Description of key information

Mammalian Cell Study: negative (Key: NTP, 1992, rel.2)

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to same study

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

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

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

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

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.

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.

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

Dose (mg/kg)	m-nitrotoluene^a
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

Conclusions:

Interpretation of results: 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

Additional information

in vitro, 3-nitrotoluene showed no mutagenic effect in Ames tests with Salmonella typhimurium, with and without metabolic activation. In cultured mammalian cells, 3-nitrotoluene has demonstrated the potential to cause mutagenicity in sister chromatid exchange test in the absence of metabolic activation. In vivo, 3-Nitrotoluene had no genotoxic activity. The substance did not did not induce unscheduled DNA synthesis in rat ex vivo hepatocytes.

Short description of key information: 

Haworth (1983)

3-nitrotoluene was tested in the Ames test similar to OECD guideline 471 with deviations (Only 4 bacterial strains were used. The highest dose tested was 10 mg/plate. 2-Aminoanthracene was tested as sole indicator of the efficacy of the S9-mix. 4-Nitro-o-phenylenediamine was tested on TA 98), using preincubation method, in strains TA98, TA100, TA1535 and TA1537 of Salmonella typhimirium.

Liver S9 fractions were prepared from male Sprague-Dawley rats and male Syrian hamsters that were injected, i.p., with Aroclor 1254. The concentrations of 3-nitrotoluene used were 0.0, 3.3, 10.0, 33.0, 100.0 and 333.0 µg/plate. The results were negative, 333.0 µg/plate resulted toxic for the strains TA 100 (with and without S9 mix), TA 1535 (without S9 mix), TA 1537 (without S9 mix) and TA 98[with (hamster) and without S9 mix].

NTP, 1992, sister chromatid exchange assay

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.

NTP, 1992, rat, in vivo, mammalian cell study

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.

Ames test conducted with a method equivalent to OECD guideline 471 yielded negative results with and without metabolic activation.
Sister chromatid exchange assay in mammalians cells carried out with a method similar to OECD guideline 479, yielded negative results with and without metabolic activation.
Unscheduled DNA Synthesis in male rats yielded negative results.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Harmonised classification:

The substance has no harmonised classification according to the Regulation (EC) No. 1272/2008 (CLP). 

 

Self classification:

Based on the available information no additional self-classification is proposed according to the CLP and to the GHS.