2-nitrotoluene

Genetic toxicity: in vivo

Administrative data

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)

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1992

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

2-nitrotoluene

EC Number:

201-853-3

EC Name:

2-nitrotoluene

Cas Number:

88-72-2

Molecular formula:

C7H7NO2

IUPAC Name:

1-methyl-2-nitrobenzene

Details on test material:

- 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

Test animals

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

Administration / exposure

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

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 200, or 500 mg/kg (male rats); 0, 200, 500, or 750 mg/kg (female rats)
Basis:
other: nominal

No. of animals per sex per dose:

12h: 3
24h: 3

Control animals:

yes, concurrent vehicle

Positive control(s):

2,6-dinitrotoluene

Examinations

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 Student¿s t-test modified for unpaired observations with unequal variances.

Results and discussion

Test results

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).

Any other information on results incl. tables

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)

Applicant's summary and conclusion

Executive summary:

NTP, 1992

 

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).