4-nitrotoluene

Genetic toxicity: in vitro

Administrative data

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)

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1987

Reference 2

Reference Type:

review article or handbook

Title:

Unnamed

Year:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 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

Test material

Test material information

Constituent 1

Reference substance name:

4-nitrotoluene

EC Number:

202-808-0

EC Name:

4-nitrotoluene

Cas Number:

99-99-0

Molecular formula:

C7H7NO2

IUPAC Name:

1-methyl-4-nitrobenzene

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

(CHO-W-B)

Metabolic activation:

with and without

Metabolic activation system:

S9 was from the livers of Aroclor 1254-induced male Sprague-Dawley rats

Test concentrations with justification for top dose:

Without S9 first 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

Controls

Controls 1

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

Controls 2

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.

Results and discussion

Test results

Test results 1

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

Test results 2

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

Any other information on results incl. tables

strain/cell type: Chinese hamster Ovary (CHO)

Applicant's summary and conclusion

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.