4-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)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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 on substance purity, cells harvested after only 1 mitotic cycle, only 100 metaphases per concentration was evaluated)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

(cells harvested after only 1 mitotic cycle, only 100 metaphases per concentration was evaluated)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

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 and second trial: 300; 400; 500 µg/ml;
Without S9 second trial: 300, 400, 500 µg/ml
With S9 second trial: 400, 500, 550 µg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Migrated to IUCLID6: -S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: +S9

Details on test system and experimental conditions:

Each concentration was test in duplicate cultures.
Doses were based on observations of cell confluence and mitotic cell availabiity in the SCE test (performed alongside chromosome aberration test). The doses for the aberration tests were usually spaced more closely than the half-log series of the preliminary SCE test and extended into the toxic range. In some tests (largely those at LBI), information on the extent of cell cycle delay seen in the SCE test was used to select a later cell harvest time for the aberration test. (Allowance was made for changes in the degree of cytotoxicity resulting from the shorter incubation time as compared with the SCE test).
The cell harvest time for the aberration test was 10.5 h after the beginning of treatment.
This yielded cells in their first mitosis. In the presence of S9, cells were incubated with onitrotoluene or solvent for 2 hours at 37° C. Cells were then washed, medium was added, and incubation was continued for 8.5 hours. Colcemid was added for the last 2 to 3 hours of incubation before harvest. In the absence of S9, cells were incubated with o-nitrotoluene or solvent for 8.5 hours at 37° C. Cells were then washed and fresh medium containing
Colcemid was added for an additional 2 hours followed by harvest. Cells were collected by mitotic shake-off.
Slides were stained with Giemsa (6%), and 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis and from positive (mitomycin or cyclophosphamide) and solvent controls. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of "simple" (breaks and terminal deletions), "complex" (exchanges and rearrangements), "other" (includes pulverized chromosomes), and "total." Gaps and endoreduplications were recorded but were not included in the totals. Aberrations in polyploid cell were not scored but used metaphases with 19-23 chromosomes (the modal number being 21)

Evaluation criteria:

Chromosomal aberration data is presented as percentage of cells with aberrations. Both the dose-response curve and individual dose points were statistically analyzed. For a single trial, a statistically significant (P<05) difference for 1 dose point and a significant trend (P<0.015) was considered weak evidence for a positive response (W+); significant differences for two or more doses indicated the trial was positive (+)

Statistics:

For chromosome aberrations, linear regression analysis of the percentage of cells with aberrations versus the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used, and the test was that described by Margolin et al [1983, pp 714-715].
The P values were adjusted by Dunnett's method to take into account the multiple dose comparisons. For data analysis, the "total" aberration category, and the criterion for a positive response was that the adjusted P value be <= 0.05.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

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:

positive

Remarks:

weak positive in the first trial and positive in the second trial

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

strain/cell type: Chinese hamster Ovary (CHO)

Executive summary:

Effects on chromosomes were studied in Chinese hamster ovary (CHO) cells in tests corresponding to the current guideline (Galloway et al., 1983; 1987; US Department of Health and Human Services, 1992; 2002). In the presence, but not in the absence of metabolic activation, 4-nitrotoluene induced chromosome damage at cytotoxic dose levels.

Reference 2

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 on substance 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 : 50; 167; 500 µg/ml;
without S9 second trial: 200, 300, 400, 500 µg/ml;
With S9 first trial: 50; 165; 500 µg/ml;
with S9 second trial: 600, 700 µg/ml;
with S9 third trial: 550, 600, 650 µg/ml

Vehicle / solvent:

DMSO

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: -S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: +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.

Statistics:

For SCE data, a linear regression trend has been used (trend test) of SCEs per chromosome vs log of the dose [Armitage, 1955. Biometrics 11:374-386]

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

weak positive in the first trial and positive in the second trial

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cell cycle delay

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

other: Second trial. weak positive [Significant increase of SCE rate of SCE/chromosome over controls in the presence of metabolic activation at highest concentration]

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

cell cycle delay

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

third trial [Significant increase of SCE rate of SCE/chromosome over controls in the presence of metabolic activation)]

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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:

first trial

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

strain/cell type: Chinese hamster Ovary (CHO)

Executive summary:

4-Nitrotoluene induced increases in sister chromatid exchange rates in Chinese Hamster Ovary (CHO) cells with and without S9-mix at doses that induced cell cycle delay, which is an indication of cytotoxicity (Galloway et al., 1987)

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

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 guideline study with acceptable restrictions (colony size not reported)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

Colony size not reported

Principles of method if other than guideline:

Protocol presented by Myhr et al., Prog. Mutat. Res. 5, 555-568 (1985), highest dose was determined by limit of solubility and toxicity, colony size not reported

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Liver S9-fraction was routinely prepared from male Sprague-Dawley rats and male Syrian hamster that were injected , i.p., with Aroclor 1254.

Test concentrations with justification for top dose:

-S9-mix: ethanol: (1) 0, 75, 100, 150, 180, 200, 240 (2) 0, 25, 50, 75, 100,
150, 250 ug/ml
+S9-mix: I. acetone: (1)(2)(3) 0, 50, 75, 100, 150, 200, 300, 500,
II. Ethanol: 50, 100, 150, 200, 250, 300 ug/ml

Vehicle / solvent:

acetone; ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: in the presence of S9-mix: methylcholantrene in the absence of S9-mix: methyl methansulfonate

Details on test system and experimental conditions:

L5178Y mouse lymphoma cells were maintained at 37° C as suspension cultures in supplemented Fischer´s medium; normal cycling time was approximately 10 hours. To reduce the number of spontaneously occurring cells resistant to trifluorothymidine (TFT), subcultures were exposed to medium containing thymidine, hypoxanthine, methotrexate, and glycine for 1 day; to medium containing thymidine, hypoxanthine, and glycine for 1 day; and to normal medium for 3 to 5 days. For cloning, the horse serum content was increased and Noble agar was added.
All treatment levels within an experiment, including concurrent positive and solvent controls, were replicated.
Treated cultures contained 6 × 106 cells in 10 mL medium. This volume included the S9 fraction in those experiments performed with metabolic activation. Incubation with p-nitrotoluene continued for 4 hours, at which time the medium plus p-nitrotoluene was removed, and the cells were resuspended in fresh medium and incubated for an additional 2 days to express the mutant phenotype. Cell density was monitored so that log phase growth was maintained. After the 48-hour expression period, cells were plated in medium and soft agar supplemented with TFT for selection of TFT-resistant cells, and cells were plated in nonselective medium and soft agar to determine cloning efficiency. Plates were incubated at 37° C in 5% CO2 for 10 to 12 days. The test was initially performed without S9. Because a clearly positive response was not obtained, the test was repeated using freshly prepared S9 from the livers of Aroclor 1254-induced male F344 rats

Evaluation criteria:

All data were evaluated statistically for trend and peak responses

Statistics:

Both responses had to be significant (P=0.05) for p-nitrotoluene to be considered positive, i.e., capable of inducing TFT resistance. A single significant response led to a call of ¿questionable,¿ and the absence of both a trend and peak response resulted in a ¿negative¿ call.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Solvent acetone: trial 1,2,3: 300 ug/ml: pracipitate of 4-nitrotoluene; 500 ug/ml: lethal; 50-200 ug/ml: significant positive response

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Solvent ethanol: Trial (1): 250, 300 ug/ml: precipitate of 4-nitrotoluene; 50, 100 ug/ml and 200, 250, 300 ug/ml: significant positive response,

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Solvent ethanol: Trial (1): 240 ug/ml: significant positive response

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no cytotoxicity or precipitation observed

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Solvent ethanol: Trial (2): 150, 250 ug/ml: significant positive response

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

Solvent ethanol: 150 ug/ml

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

strain/cell type: mouse lymphoma L5178Y cells

Executive summary:

4-Nitrotoluene was tested positive in a L5178Y mouse lymphoma assay in the presence of rat liver S9 mix. As the colony size is not reported, it is unclear whether this response can be attributed to gene mutations or cytogenetic effects (U.S. Department of Health and Human Services, 1992; 2002).

Reference 4

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 (only four strains)

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:

0, 3.3, 10.0, 33.0, 100.0, 333.3, 500.0 ug/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

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 (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 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 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 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.

TA 100

TA 1535

TA 1537

TA 98

Dose

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

0.0

127±13.3

128±15.3

145±8.7

22±2.0

9±0.3

11±2.1

5±1.9

7±1.7

4±1.5

13±1.7

23±5.6

23±2.1

3.3

125±10.5

24±0.9

4±2.6

11±0.6

10.0

141±5.3

118±9.5

131±3.8

20±1.3

9±1.9

15±0.7

5±0.9

7±1.9

7±0.7

15±1.7

23±2.3

23±2.5

33.3

109±6.5

131±3.9

134±6.9

19±1.2

14±2.5

10±1.0

4±1.5

5±2.0

6±0.3

15±1.2

20±1.2

22±0.3

100.0

124±9.8

137±6.2

136±5.7

19±3.0

11±1.7

12±2.2

3±1.2

6±1.0

7±0.0

14±1.3

24±4.1

27±4.5

333.3

147±3.8s

137±4.5

164±5.5

21±2.7s

16±1.5

17±1.9

4±0.7

6±2.7

5±2.5

11±2.0

22±0.9

27±1.2

500.0

136±5.0s

143±11.9s

10±1.5s

11±2.6s

6±1.0s

6±1.0s

16±2.0s

27±5.0s

POS

1343±30.9

1061±52.5

1191±27.7

980±33.6

82±3.5

49±2.5

379±61.2

77±6.1

133±3.8

1347±43.7

752±10.7

1045±12.6

 

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

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

Executive summary:

Haworth (1983):

 

Ames test was performed with the Salmonella typhimuriumstrains TA1535, TA1537, TA98, and TA100 at 0, 3.3, 10, 33.3, 100, 333.3, and 500 µg 4 -nitrotoluene/plate with and without S-9 from Aroclor 1254 induced rat and hamster livers using the preincubation procedure. Test doses were chosen following checks for toxicity: in the absence of toxicity a maximum of 10 mg/plate was used. The result was negative.

The test was conducted according to OECD guideline 471 with deviations (Only 4 strains tested instead of 5. Positive control: 2-aminoantracene was only indicator of efficiency of S9-mix. Individual-plate-readings were not reported.)

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro, 4-nitrotoluene showed no mutagenic effect in good quality Ames tests with Salmonella typhimurium and Escherichia coli, with and without metabolic activation. In cultured mammalian cells, 4-nitrotoluene has demonstrated the potential to cause mutagenicity in the presence of metabolic activation. The chemical did not induced unscheduled DNA synthesis in hepatocytes.

 

In vivo, 4-Nitrotoluene had no genotoxic activity. The substance did not induce micronuclei in rat and mice bone marrow cells in studies performed according to the current standard (OECD TG 474) and it did not induce unscheduled DNA synthesis in rat ex vivo hepatocytes.

Short description of key information: sourced from OECD SIDS

In vitro Studies
In tests performed according to current standards, 4-nitrotoluene was not mutagenic in Salmonella typhimurium strains TA98, TA100, TA1535, TA1537, TA102, TA104 and in Escherichia coli
WP2uvra (dose-range, with and without metabolic activation: 0.0763 - 5000 µg/plate (JETOC 1996); 3.3 - 1000 µg/plate (U.S. Department of Health and Human Services, 2002). The studies gave no indication of gene mutation with and without metabolic activation. The positive controls were functional.

In earlier studies also some positive responses were obtained, mainly in Salmonella typhimurium
TA100. These results, however, were either not well documented, or were obtained with nonspecified test material or in non-standard test systems (e.g. Shimizu and Yano, 1986; Nohmi et al., 1984; Melnikov et al., 1981). These positive findings were therefore considered as of less relevance as compared to the data from tests performed in accordance with current standards. Overall, based on the studies of best quality, 4-nitrotoluene is considered as non-mutagenic in the Ames test system.
4-Nitrotoluene was tested positive in a L5178Y mouse lymphoma assay in the presence of rat liver
S9 mix. As the colony size is not reported, it is unclear whether this response can be attributed to gene mutations or cytogenetic effects (U.S. Department of Health and Human Services, 1992;
2002).

Effects on chromosomes were studied in Chinese hamster ovary (CHO) cells in tests corresponding to the current guideline (Galloway et al., 1983; 1987; US Department of Health and Human Services, 1992; 2002). In the presence, but not in the absence of metabolic activation, 4-nitrotoluene induced chromosome damage at cytotoxic dose levels.

4-Nitrotoluene induced increases in sister chromatid exchange rates in Chinese Hamster Ovary
(CHO) cells with and without S9-mix at doses that induced cell cycle delay, which is an indication of cytotoxicity (Galloway et al., 1987; US Department of Health and Services, 1992; 2002).

 

Indicator test: 4-nitrotoluene caused no unscheduled DNA synthesis (UDS) in cultured primary hepatocytes of rats (Doolittle et al., 1983).

 

Conclusion

In vitro, 4-nitrotoluene showed no mutagenic effect in good quality Ames tests with Salmonella typhimurium and Escherichia coli, with and without metabolic activation. In cultured mammalian cells, 4-nitrotoluene has demonstrated the potential to cause mutagenicity in the presence of metabolic activation. The chemical did not induced unscheduled DNA synthesis in hepatocytes.

 

In vivo Studies

There are bone marrow micronucleus tests available, that were performed with male rats and male mice according to OECD TG 474. 4-Nitrotoluene caused no increases in micronucleated polychromatic erythrocytes (PCEs) in the bone marrow of male rats given 0, 150, 300, or 600 mg/kg bw by intraperitoneal injection three times at 24 hour intervals. In male mice, treated with 0, 150, 300, or 600 mg/kg bw by intraperitoneal injection three times at 24 hour intervals, results of a first trial were considered positive, based on the responses of the two lowest doses; the trend test was not significant due to a downturn at the highest dose level. A second trial failed to induce a significant increase in micronucleated PCEs over the same dose range. Therefore the authors concluded the overall results as negative. Neither in the study with rats nor in the studies with mice were any signs of toxicity reported (U.S. Department of Health and Human Services, 2002). Indicator test 4-Nitrotoluene did not induce unscheduled DNA synthesis (UDS) in hepatocytes of rats after single oral treatment by gavage with 0, 100, 200, 500 mg/kg bw dissolved in corn oil or 0, 50, 200, 1000 mg/kg bw in corn oil (US Department of Health and Human Services, 1992; Mirsalis, 1989).

 

Conclusion

In vivo, 4-Nitrotoluene had no genotoxic activity. The substance did not induce micronuclei in rat and mice bone marrow cells in studies performed according to the current standard (OECD TG 474) and it did not induce unscheduled DNA synthesis in rat ex vivo hepatocytes.

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.