Nitrotoluene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is no repeated dose toxicity study available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from 2-nitrotoluene (CAS 88-72-2).

2-NT is negative in Ames Assay (Haworth et al. 1983).
2-NT is positive in Mouse in in vitro Micronucleus Assay (Matsushima 1999).
2-NT is resulting in sister chromatid exchanges in presence of metabolic activation (Galloway 1987).
Endpoint Conclusion: Adverse effect observed (positive).

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

Specific details on test material used for the study:

- Name of test material (as cited in study report): o-nitrotoluene
- Analytical purity: no data

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: 117; 176; 218; 282 µg/ml
With S9: 354.83; 380.95; 423.28 µg/ml

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.

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

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

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

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.

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

Specific details on test material used for the study:

- Name of test material (as cited in study report): o-nitrotoluene
- Analytical purity: > 99%
- Lot Number: A8A

Target gene:

his

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

microsomal enzyme reaction mix (S9-mix).
Liver S9 fractions were prepared from male Sprague-Dawley rats and male Syrian hamsters that were injected, i.p., with Aroclor 1254.

Test concentrations with justification for top dose:

SRI (LABORATORY): 0; 3.0; 10.0; 33.0; 100.0; 333.0; 666.0 µg/plate
EGG (LABORATORY): 0; 3.3; 10.0; 33.0; 100.0; 333.0µg/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:

9-aminoacridine

sodium azide

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.

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

True negative controls validity:

not applicable

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

True negative controls validity:

not applicable

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

True negative controls validity:

not applicable

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

True negative controls validity:

not applicable

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.

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

TA 100

TA 1535

TA 1537

TA 98

Dose

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

0.0

138±16.3

123±7.9

134±6.2

25±3.8

10±0.6

14±1.7

8±1.5

10±1.5

13±1.2

14±2.7

25±0.7

28±1.2

3.3

135±4.3

149±7.8

127±2.3

26±2.5

10±1.2

15±3.2

8±2.1

8±0.9

9±2.1

20±3.8

29±1.2

31±1.9

10.0

139±5.8

128±3.2

118±7.6

23±3.3

9±1.7

17±2.5

5±1.8

6±1.2

12±0.0

16±1.9

27±0.3

31±7.1

33.3

122±6.9

147±21.7

134±2.5

24±1.7

10±3.3

20±1.5

6±1.2

7±2.1

9±0.6

22±1.2

27±5.2

32±1.9

100.0

121±11.1

142±5.2

135±9.3

22±4.9

19±2.0

16±1.5

8±1.3

6±0.9

8±1.2

20±1.2

25±1.9

34±2.3

333.3

132±9.2s

115±11.0

148±3.3

23±2.8s

12±1.2

16±2.5

6±0.3s

7±0.9

11±0.9

12±1.8s

27±3.7

36±3.5

POS

2103±44.5

991±44.5

1900±92.3

1320±43.1

108±5.5

128±6.3

901±105.9

71±4.9

173±7.2

2119±51.4

874±29.2

1454±95.4

Abbreviations: NA, not activated; RLI, rat liver S-9, Aroclor1254induced; HLI, hamster liver S-9,Aroclor1254 induced, t=complete clearing background

 

 

Table 2 Mutagenic responses of Salmonella strains TA100, TA 1535, TA 1537, and TA 98 (mean±SEM) to o-nitrotoluene.

TA 100

TA 1535

TA 1537

TA 98

Dose

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

NA

RLI

HLI

0.0

120±4.7

117±7.8

126±9.1

15±1.7

10±1.9

12±2.1

5±2.6

6±0.9

8±2.6

22±2.7

24±2.6

28±4.6

3.3

118±8.5

10±1.5

4±0.6

24±1.9

10.0

105±4.7

130±3.8

133±4.7

11±1.2

10±2.8

6±1.7

3±0.7

5±1.3

9±2.3

16±0.9

29±2.3

35±1.7

33.3

110±7.5

133±7.5

125±0.9

14±1.5

10±2.1

10±2.6

4±0.3

6±0.7

6±1.2

17±1.5

30±0.9

29±1.8

100.0

104±11.2

147±5.3

125±13.9

18±5.2

7±2.9

10±1.5

3±0.9

8±0.7

8±2.6

36±22.3

34±2.7

27±1.3

333.3

t

114±1.9

125±7.0

0±0

11±1.5

8±0.7

t

6±1.0

6±1.5

0±0.0s

31±3.8

28±2.6

666.0

119±2.5

137±5.7

9±3.0

t

6±1.3

t

31±0.5

0±0.0s

POS

424±16.2

900±15.3

1895±83.7

396±2.3

313±77.0

507±35.4

100±18.2

283±14.2

353±32.0

760±8.0

640±8.7

1761±147.7

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

 

 

 

Executive summary:

2-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, 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 2-nitrotoluene used were 0.0, 3.3, 10.0, 33.0, 100.0 and 333.0µg/plate in one experiment (EGG laboratory) and

0.0, 3.3, 10.0, 33.0, 100.0, 333.0, and 666.0 µg/plate (SRI laboratory).

The results were negative, 333.0µg/plate resulted toxic for the strains TA 100, TA 1537 and TA 98 without S9 mix.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus 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 GLP status, no data on substance purity, no trial performed with S9 mix, cytotoxicity and CBPI not reported)

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Draft Guideline 487 (Genetic Toxicology: In Vitro Micronucleus Test)

Deviations:

yes

Remarks:

(no trial performed with S9 mix, no data on substance purity given, cytotoxicity and CBPI not reported)

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

CHL/lU cells obtained from the Japanese Collection of Research Bioresources (JCRB) were used throughout the collaborative study. They were maintained in Eagle's minimum essential medium supplemented with 10 % heat inactivated (56°C for 30 min) calf serum.

Metabolic activation:

with and without

Metabolic activation system:

co-factor-supplemented postmitochondrial fraction (S9) prepared from phenobarbital- and 5,6-benzoflavone-pretreated male Sprague-Dawley rats

Test concentrations with justification for top dose:

2-50 µg/ml

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Positive control Cyclophosphamide and benzo[a]pyrene (with S9 mix), Mitomycin C (without S9 mix)

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

without S9 mix; max frequency MN/1000 cells = 56 MN versus 5 MN in control)

Cytotoxicity / choice of top concentrations:

not specified

Executive summary:

o-Nitrotoluene was positive in the micronucleus test after treatment of CHL/lU cells with 2 -50 µg/ml 2 -nitrotoluene for 6h with an adjoining 18h recovery period. Max frequency of micronucleus per 1000 cell was 56 MN for assays without metabolic activation. For control cells (DMSO), 5 MN/1000 were noted.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

There is no experimental test data available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from its components. Nitrotoluene (CAS 1321-12-6) is a mixture of mainly 4-nitrotoluene (CAS 99-99-0) and/or 2-nitrotoluene (CAS 88-72-2). In addition, the mixture is containing small amounts of 3-nitrotoluene (CAS 99-08-1). A wealth of data is existing for the hazard assessment of 2- and 4-nitrotoluene, which can be used for the classification of nitrotoluene (CAS 1321-12-6). Key data and classification are derived from the isomer with the most critical hazard identified for each specific end point. The available experimental test data are considered reliable and suitable for the classification of nitrotoluene (CAS 1321-12-6) under Regulation 1272/2008.

Reason / purpose for cross-reference:

read-across source

Type of assay:

bacterial reverse mutation assay

Metabolic activation:

with and without

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not applicable

Positive controls validity:

valid

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

There is no experimental test data available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from its components. Nitrotoluene (CAS 1321-12-6) is a mixture of mainly 4-nitrotoluene (CAS 99-99-0) and/or 2-nitrotoluene (CAS 88-72-2). In addition, the mixture is containing small amounts of 3-nitrotoluene (CAS 99-08-1). A wealth of data is existing for the hazard assessment of 2- and 4-nitrotoluene, which can be used for the classification of nitrotoluene (CAS 1321-12-6). Key data and classification are derived from the isomer with the most critical hazard identified for each specific end point. The available experimental test data are considered reliable and suitable for the classification of nitrotoluene (CAS 1321-12-6) under Regulation 1272/2008.

Reason / purpose for cross-reference:

read-across source

Type of assay:

in vitro mammalian cell micronucleus test

Metabolic activation:

with and without

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Reference 6

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

There is no experimental test data available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from its components. Nitrotoluene (CAS 1321-12-6) is a mixture of mainly 4-nitrotoluene (CAS 99-99-0) and/or 2-nitrotoluene (CAS 88-72-2). In addition, the mixture is containing small amounts of 3-nitrotoluene (CAS 99-08-1). A wealth of data is existing for the hazard assessment of 2- and 4-nitrotoluene, which can be used for the classification of nitrotoluene (CAS 1321-12-6). Key data and classification are derived from the isomer with the most critical hazard identified for each specific end point. The available experimental test data are considered reliable and suitable for the classification of nitrotoluene (CAS 1321-12-6) under Regulation 1272/2008.

Reason / purpose for cross-reference:

read-across source

Type of assay:

sister chromatid exchange assay in mammalian cells

Metabolic activation:

with and without

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

There is no repeated dose toxicity study available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from 2-nitrotoluene (CAS 88-72-2).

2-NT is positive in an OECD486 Unsheduled DNA Synthesis Test in liver cells from gavaged rats (NTP 1992).
Endpoint Conclusion: Adverse effect observed (positive).

Link to relevant study records

Reference

Reference 1

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: Comparable to guideline with acceptable restriction (only 90 cells scored /dose, results of the positive control not given, postive control not guideline listed and no rational for choice is given)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

yes

Remarks:

total of 90 cells scored/ dose, positive control not listed in guideline

Principles of method if other than guideline:

According to the method of Mirsalis et al (1985) Carcinogenesis 6, 1521-1524

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

- Source: Aldrich Chemical Co. (Milwaukee, WI, USA),
- Analytical purity: >96%
- Impurities: < 1% (mostly m- and p-nitrotoluene)
- Storage: RT
- Stability: reanalysis performed at approx. 4 months intervals indicated that the test substance was stable under the storage conditions chosen

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Taconic Farms, Inc. (Germantown, NY
- Age at study initiation: 11 to 12 weeks
- Housing: 3/cage
- Acclimation period: 7-8 weeks
OTHER: Assignment to treatment groups; by weight class using a computer-generated randomization procedure.

ENVIRONMENTAL CONDITIONS: not reported

Route of administration:

oral: gavage

Vehicle:

Vehicle: Corn oil

Details on exposure:

DOSE VOLUME: 5 ml/kg bw

Duration of treatment / exposure:

12, 24h

Frequency of treatment:

single application

Post exposure period:

no

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

males

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

males

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

males

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

females

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

females

Dose / conc.:

750 mg/kg bw/day (nominal)

Remarks:

females

No. of animals per sex per dose:

12h: 3
24h: 3

Control animals:

yes, concurrent vehicle

Positive control(s):

2,6-dinitrotoluene

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: no data
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): 12h and 24h after gavage, 3 rats were selected from each group for the collection of hepatocytes for UDS determination. 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.
DETAILS OF SLIDE PREPARATION:
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 for 4h. Cell cultures were found to contain >80% viable cells. 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 after which they were washed (Williams Medium E without serum) and the nuclei swollen by the incubation with 1% sodium citrate (8-12min) and fixed in glacial acetic acid: ethanol (1:3), washed (deionized water) and dried for for at least 24h. 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.
METHOD OF ANALYSIS:
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 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).

Evaluation criteria:

According to the srtudy report, 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 examin d 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.

Statistics:

Significance of the response was determined using the Students t-test modified for unpaired observations with unequal variances.

Sex:

male/female

Genotoxicity:

positive

Remarks:

Statistically significant increase of UDS from 100 mg/kg bw in male rats and from 200 mg/kg bw in female rats. Also an increase of the number of hepatocytes in S-phase, cultured from both male (at 500 mg/kg bw) and female (>= 200 mg/kg bw) rats was seen

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

Along o-nitrotoluene, m-, and p- nitrotoluene were also included in the panel of substances tested in the rat in vivo/in vitro UDS test. A comparison of UDS activity of the 3 nitrotoluene isomers was performed using identical doses (0, 100, 200, 500 mg/kg bw) given to male F344 rats. From these data, it was apparent that the o-nitrotoluene was the only isomer which was positive for induction of UDS.
o-Nitrotoluene was also found to increase the number of hepatocytes in S-phase, cultured from both male and female rats (see table 1). In this assay, neither m- or p-nitrotoluene caused an increase in S-phase hepatocytes in either sex of the rats (data not shown in documentation).

Table 1:Unscheduled DNA Synthesis in Male and Female Rats and Percent Liver Cells in S-Phase (12 and 24 Hours after Single Oral Gavage Dose of o-Nitrotoluene, respectively)

Dose	Male		Female
mg/kg bw	UDS (12h after gavage)	Cells in S phase (24 h after gavage)	UDS (12h after gavage)	Cells in S phase (24 h after gavage
	mean net nuclear grain count ± SD	%	mean net nuclear grain count ± SD	%
0	-2.57 ± 0.18	0.66 ± 0.18	-5.96 ± 0.59	0.58 ± 0.27
100	- 0.05 ± 0.47*	0.86 ± 0.42	Not tested	Not tested
200	5.64 ± 0.57**	3.61 ± 0.94	- 2.24 ± 1.0**	2.40 ± 0.70
500	13.11 ± 1.14**	3.20 ± 0.47*	0.94 ± 0.93**	7.17 ± 0.70
750	Not tested	Not tested	1.45 ± 0.93*	12.98 ± 3.9

* Significantly different from control group (P=0.05)

** Significantly different from control group (P=0.01)

Executive summary:

Groups of 6 male F344/ rats/dose and 6 female F344 rats/dose were administered oral doses of o-Nitrotoluene in corn oil via gavage; 0; 100; 200; 500 mg/kg for males and 0; 200; 500 and 750 mg/kg bw for females, respectively, according to a method similar to OECD guideline 486 (total of 90 cells scored/ dose, positive control not listed in guideline) Perfusion of liver and preparation of hepatocytes was performed 12h and 24h after gavage.

o-Nitrotoluene induced DNA damage in mammalian liver cells in vivo (male and female F344 rats) that could be repaired by unscheduled DNA synthesis in vitro; Male rats (>= 100 mg/kg bw), Female rats (>= 200 mg/kg bw).

 

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

There is no experimental test data available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from its components. Nitrotoluene (CAS 1321-12-6) is a mixture of mainly 4-nitrotoluene (CAS 99-99-0) and/or 2-nitrotoluene (CAS 88-72-2). In addition, the mixture is containing small amounts of 3-nitrotoluene (CAS 99-08-1). A wealth of data is existing for the hazard assessment of 2- and 4-nitrotoluene, which can be used for the classification of nitrotoluene (CAS 1321-12-6). Key data and classification are derived from the isomer with the most critical hazard identified for each specific end point. The available experimental test data are considered reliable and suitable for the classification of nitrotoluene (CAS 1321-12-6) under Regulation 1272/2008.

Reason / purpose for cross-reference:

read-across source

Type of assay:

unscheduled DNA synthesis

Species:

rat

Sex:

male/female

Genotoxicity:

positive

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

not specified

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

Mononitrotolueneis a mixture of mainly 4-nitrotoluene (CAS 99-99-0) and/or 2-nitrotoluene (CAS 88-72-2). In addition the mixture is containing small amounts of 3-nitrotoluene (CAS 99-08-1). A wealth of data is existing for the hazard assessment of 2- and 4-nitrotoluene. Key data and classification is derived from the isomer with the most critical hazard identified for each specific end point. For completeness key data of the other isomer is added as supporting information.

Regarding genetic toxicity classification is driven by 2-nitrotoluene.

Genetic toxicity of 2 -nitrotoluene (incomplete):

There are several data available on the genotoxicity of 2-nitrotoluene in vitro and in vivo studies.

The information in bacteria indicates that 2-nitrotoluene is not mutagenic in any of several strains of Salmonella typhimurium with or without metabolic activation enzymes (Haworth 1983).

In cytogenetic tests on Chinese Hamster Ovary cells, 2-nitrotoluene increased the Sister Chromatid Exchange rate, being more pronounced in the presence of S9 mix (Galloway 1987).

There was an increase in polyploidy cells when 2-nitrotoluene was tested in cultures of Chinese hamster lung (CHL) cells in the absence of S9 mix (Matsushima 1999).

Positive results were found in the UDS test for both male and female rats administered 2-nitrotoluene (NTP 1992).

In conclusion, 2-nitrotoluene is mutagenic in somatic cells. In addition, it reaches the germ cells since toxicity was observed in testis and epididymis.

Genetic toxicity of 4 -nitrotoluene (incomplete):

Similarly to 2-NT, no mutagenicity was identifierd with 4-NT in the Ames Assay (Haworth 1983). Though indications of a mild genotoxic potential were identified in a Mouse Lymphoma Assay in absence of a metabolising system (NTP 1992) and in a Chromosomal Aberration Assay at cytotoxic concentrations in presence of a metabolising system (Galloway 1983).

However for 4-NT these indications of a genotoxic potential from in vitro test systems were not substantiated in an in vivo Micronucleus Assay in which rats were intraperitoneally exposed (NTP 2001).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. There is no in vitro or in vivo genotoxicity study available for nitrotoluene (CAS 1321-12-6), but a read-across can be made from 2-nitrotoluene (CAS 88-72-2). 2-nitrotoluene was tested positive in various in vitro and in vivo genotoxicity tests.

Furthermore, 2-nitrotoluene (CAS 88-72-2) is included in Annex VI of Regulation (EC) No. 1272/2008 with the following classification:

• Muta. Cat. 1B (H340)

Therefore, nitrotoluene (CAS 1321 -12 -6) is also classified for Muta. Cat. 1B (H340: May cause genetic effects) under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.