1,2-dichloro-4-nitrobenzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1,2 -dichloro-4 -nitrobenzene was tested in the Ames test according to OECD TG 471 with and without a metabolic activation system and up to cytotoxicity, yielded a positive result in strain S. typhimurium TA 100 (JETOC 2005).

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).

Link to relevant study records

Reference

Reference 1

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

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

Target gene:

HGPRT

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

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

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

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).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial gene mutation assay

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

S. typhimurium TA 1537

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

liver S9-fraction from male Sprague-Dawley rats induced by sodium phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

trial 1: +/- S9-mix: 1.22, 4.88, 19.5, 78.1, 313, 1250, 5000 µg/plate
trial 2: +/-S9-mix: 4.88, 9.77, 19.5, 39.1, 78.1, 156, 313 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

furylfuramide

other: 2- Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation;
DETERMINATION OF TOXICITY: growth inhibition

Evaluation criteria:

Two-hold rule criteria was used for data evaluation (Ames et al 1975). The chemicals were considered to be mutagenic when a dose related increase in revertant colony count was observed and the number of revertant colonies per plate with the test substance was more than twice that of the negative control and when a reproducibility of the test result was observed.

Statistics:

see above

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:

valid

True 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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

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:

valid

True 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:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

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

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Conclusions:

1,2 -dichloro-4 -nitrobenzene yielded a positive result only in Salmonella typhimurium TA 100 in the presence and in the absence of an additional metabolic activation system. S. Typhimurium TA 98, TA1535, TA1537 and E.Coli WPurvA/pKM101 yielded negative results in the presence and in the absence of an additional metabolic activation system.

Executive summary:

1,2 -dichloro-4 -nitrobenzene, tested in the Ames test according to OECD TG 471 with and without a metabolic activation system and up to cytotoxicity and yielded a positive result only in Salmonella typhimurium TA 100 in the presence and in the absence of an additional metabolic activation system. S. Typhimurium TA 98, TA1535, TA1537 and E.Coli WPurvA/pKM101 yielded negative results in the presence and in the absence of an additional metabolic activation system (JETOC 2005).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

1,2-dichloro-4 -nitrobenzene was tested in a mammalian erthrocyte micronucleus test in ICR mice and yielded a negative result.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

only one treatment and one sampling after 24 h (no 2nd sampling after 48 h)

Deviations:

yes

Remarks:

one treatment only and only one sampling after 24 h (no 2nd sampling after 48 h)

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Manufactured by Tokio Kasei Kogyo Co. Ltd., Japan

Species:

mouse

Strain:

ICL-ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Dae-Han Laboratory Animal Co., Eumsunggun Korea
- Age at study initiation: 7-8 weeks old
- Assigned to test groups randomly: yes
- Housing: six animals were housed for each group
- Diet (e.g. ad libitum): commercial pellets ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 1 week

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)
- Amount of vehicle (if gavage or dermal): 10mL/kg

Duration of treatment / exposure:

The test substance was given once

Frequency of treatment:

Once

Post exposure period:

24 h

Dose / conc.:

692 mg/kg bw/day (nominal)

Dose / conc.:

346 mg/kg bw/day (nominal)

Dose / conc.:

173 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C, 2 mg/kg, i.p.

Tissues and cell types examined:

bone marrow from both femora

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The rationale for the dose selection was the selection of half of the LD50 value as highest dose. The LD50 value from RTECS database for mice was 1384 mg/kg bw.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): From the freshly killed animal both femora 24 h after administration were removed in toto, which means that one was cutting through pelvis and tibia. The bones were then freed from muscle by the use of gauze and fingers. With the needle of appropriate size mounted, about 1mL of serum was pulled from the tube into a disposable plastic syringe. Then the needle (24 gauge) was inserted a few mm into the proximal part of marrow canal to flush the marrow cells.

DETAILS OF SLIDE PREPARATION: After centrifugation, the supernatant was removed, and cell pellet suspension of bone marrow cells was dropped onto glass slides, and then air dried. After fixation in methanol, slides were stained with 4% Giemsa in 1/15M sodium phosphate buffered saline (PBS, pH 6.8) for 30 min, washed with PBS, and then air dried for microscopic observation.

METHOD OF ANALYSIS: In scoring the preparations, micronuclei were counted in polychromatic and separately in normochromatic erythrocytes. The rate of micronucleated cells, expressed in percentage, were based on the total of polychromatic erythrocytes present in the scored optic fields. This mode of scoring, which must always be followed where the test substance markedly influences the proliferation rate in the bone marrow, prevents a distortion of the results by the influx of peripheral blood into the damaged marrow. The scoring of micronucleated normocytes not only serves to recognize the presence of artifacts (which is rare in preparations from mouse) but provides additional interesting information on the mode of action of the test substance. Generally, an incidence of more than 1 micronucleated normocyte per thousand polychromatic erythrocytes indicates an effect on cell stages past the S-phase.

Statistics:

pariwise comparison to corresponding control

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

no decreased no. of immature erythrocytes observed, clinical signs of toxicity not described

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Test chemicals	Dose (mg/kg)	Route	Sampling time (hr)	MNPCE%/PCE (Mean ± SD)	Ratio % of PCE/PCE+NCE (Mean ± SD)	p-value
Negative control	-	i.p	24	0.11±0.07	0.49±0.02	-
	-	p.o	24	0.14±0.08	0.50±0.1	-
Positive control (Mitocycin C)	2	i.p	24	3.42±0.79	0.50±0.01	0.0000
1,2-dichloro 4-nitrobenzene (99-54-7)	692	p.o	24	0.17±0.06	0.48±0.02	>0.05
	346		24	0.12±0.08	0.45±0.07	>0.05
	173		24	0.13±0.16	0.43±0.03	>0.05

pariwise comparison to corresponding control, significant at P < 0.05

MNPCE%/PCE: percentage of Micronucleuated polychromatic erythrocytes/1,000 polychromatic erythrocytes

PCE/PCE+NCE: polychromatic erythrocytes/1,000 erthrocytes

Conclusions:

No significant induction ratio of percentage of micronucleated polychromatic erythrocytes/1,000 poly chromatic erythrocytes (MNPCE &/ PCE) compared to solvent control.

Executive summary:

Bone marrow micronucleus assay was performed in mice. About 7-8 weeks old male ICR mice were exposed to the test item via oral gavage. Doses of 692, 346 and 173 mg/kg bw/d were applied. The rationale for the dose selection was the selection of half of the LD50 value as highest dose. The LD50 value from RTECS database for mice was 1384 mg/kg bw. 24 h after a single substance administration, the animals were killed and the bone marrow from the femurs was analysed. No significant induction ratio of percentage of micronucleated polychromatic erythrocytes/1,000 polychromatic erythrocytes (MNPCE &/ PCE) compared to solvent control was observed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

in vitro

1,2 -dichloro-4 -nitrobenzene, tested in the Ames test according to OECD TG 471 with and without a metabolic activation system and up to cytotoxicity, yielded a positive result in strain S. typhimurium TA 100 with and without metabolic activation (JETOC 2005). 1,2 -dichloro-4 -nitrobenzene technical grade was tested in the CHO/HPRT Mammalian cell forward gene mutation assay and yielded negative result (Monsonto Co 1986). Thus ist could be shown that the positive result in an bacterial mutation test could not be confirmed in an gene mutation test using a mammalian cell system.

1,2-Dichloro-4-nitrobenzene induced chromosomal aberations in CHL/IU cells when tested according to the respective guideline up to cytotoxicity (JETOC 2005).

in vivo

Bone marrow micronucleus assay was performed in mice. About 7-8 weeks old male ICR mice were exposed to the test item via oral gavage. Doses of 692, 346 and 173 mg/kg bw/d were applied. The rationale for the dose selection was the selection of half of the LD50 value as highest dose. The LD50 value from RTECS database for mice was 1384 mg/kg bw. 24 h after a single substance administration, the animals were killed and the bone marrow from the femurs was analysed. No significant induction ratio of percentage of micronucleated polychromatic erythrocytes/1,000 polychromatic erythrocytes (MNPCE &/ PCE) compared to solvent control was observed (Ryu, 2004).

1,2-dichloro-4 -nitrobenzene technical grade showed no clastogenic activity in vivo in a chromosomal aberration test in bone marrow of rats (Monsanto Co 1983).

Justification for classification or non-classification

Based on the above discussed experimental results, the substance is not classified in accordance with Regulation (EC) No 1272/2008.