1,2-dichloro-4-nitrobenzene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific priciples, acceptable for assessment, test substance techn. grade

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

-addition of different concentrations of the metabolic activation system

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,2-dichloro-4-nitrobenzene

EC Number:

202-764-2

EC Name:

1,2-dichloro-4-nitrobenzene

Cas Number:

99-54-7

Molecular formula:

C6H3Cl2NO2

IUPAC Name:

1,2-dichloro-4-nitrobenzene

Constituent 2

Reference substance name:

1,2-dichloro-3-nitrobenzene

EC Number:

221-717-7

EC Name:

1,2-dichloro-3-nitrobenzene

Cas Number:

3209-22-1

Molecular formula:

C6H3Cl2NO2

IUPAC Name:

1,2-dichloro-3-nitrobenzene

Method

Target gene:

HGPRT

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1BH4

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: CHO-K1-BH4, Dr. Abraham W. Hsie, Biology Division, Oak Ridge National Laboratories, P.O. Box Y, Oak Ridge, Tennessee 37380
- Suitability of cells: Chemicals capable of inducing mutations have been shown to incraase the
forward mutation frequency at the hgprt loclus in Chinese Hamster Ovary Cells

The stock cultures of CHO-K1-BH4 cell line are maintained in frozen aliquots in a Revco Ultra-low Freezer. Cultures of CHO-K1-BH4 cell line were prepared from stock cultures known to have a stable spontaneous mutation frequency of 0 - 10 x 10E-6 mutants per cell, however, values up to 20 x 10E-6 were deemed acceptable.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix: 1 %, 2 %, 5%, 10%

Test concentrations with justification for top dose:

1st experiment: -S9-mix: 100, 120, 150 µg/mL, +S9-mix (1, 2, 5, 10%): 50, 150, 200 µg/mL
2nd experiment: 0, 25, 50, 125, 200, 250 µg/mL (cytotoxicity of approximately 92, 70, 43, 30 and 15 % mean relative survival without metabolic activation and 81, 71, 54, 42 and 7 % mean relative survival with metabolic activation, respectively.)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate (1st experiment), triplicate (2nd experiment)
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 5 x 10E5 cells in 5 ml of medium
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 5 h
- Harvest time after the end of treatment (sampling/recovery times): 19 h

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7 - 8 d
- Selection time (if incubation with a selective agent): 7 d
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: For mutant selection, 6.25 mL of 10E-3 M 6-thioguanine solution was added to 494 mL of hypoxanthine free medium. To each of 5-100 mm plates 8 mL of the 6-TG medium were added and 2 mL of the 1 x 10 E5 cells/mL aliquot, for a total of 2 x 10E5 cells/plate. The plates were incubated for 7 days at 37°C in 5 % CO2 in air at 90+ % humidity to allow for colony formation.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative survival (RS), cloning efficiency

Evaluation criteria:

positive: mutation frequency significant greater than control, in one concentration and mean survival of at least 10 %, dose response relationship
negative: none mutation frequency greater than the of the solvent control, no dose-response relationship

Statistics:

one-way analysis of variance method outlined by Snee and Irr (1981) one-tailed student's t-test using pooled , intergroup variance; dose-response relationship

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES (if applicable):
1. toxicity test prior to testing:
dose levels:
0.33, 1.0, 3.3, 10, 33.3, 100, 333, 1000 µg/ml in the presence of 0, 1, 2, 5, and 10 % Ariclor1254 induced rat liver S9-mix
incubation time: 5 hours
result:
1000 µg/ml: cytotoxicity at all concentrations of S9-mix
333 µg/ml: reduced relative cell survival (0-10% S9): 36%, 36% 34%, 38%, 18%
2. preliminary mutagenicity screen:
dose levels:
-S9-mix: 100, 120, 150 µg/ml
+S9-mix (1, 2, 5, 10%): 50, 150, 200 µg/ml
incubation time: 5 hours with TS and after removal of TS for 19 hours and after washing for additional 7 days
result:
survival:
-S9-mix: 99, 94, 47 %
+S9-mix: 1%: 79/45/10% survival; 2%: 86/48/25% survival; 5%: 91/73/ 42% survival; 10%: 99/80/78% survival
there were no significant increases in the mutation levels when compared to the negative controls

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
Mean mutation frequency (with S9-mix - without S9-mix):
-negative controls:
untreated controls: 0.6 - 1.9
DMSO- control: 0.8 - 0.6
-positive controls:
EMS: 275 (without S9-mix)
DMN: 265 (with S9-mix)

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency:
relative cell survival (low to high dose):
-S9-mix: 92, 70, 43, 30, 15 %
+S9-mix: 81, 71, 54, 42, 7 %
mean mutation frequency (low to high dose: with/without S9-mix):
0.0/1.4, 1.3/1.1, 0.8/0.5, 0.6/1.5, 1.0/1.4
->no statistically significant difference when compared to the negative controls

Applicant's summary and conclusion

Conclusions:

1,2 -dichloro-4 -nitrobenzene technical grade was tested negative in this CHO/HPRT Mammalian cell forward gene mutation assay.

Executive summary:

1,2 -dichloro-4 -nitrobenzene technical grade was tested in the CHO/HPRT Mammalian cell forward gene mutation assay and yielded negative result (Monsanto Co 1986).