1-chloro-2-nitrobenzene

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1-chloro-2-nitrobenzene

EC Number:

201-854-9

EC Name:

1-chloro-2-nitrobenzene

Cas Number:

88-73-3

Molecular formula:

C6H4ClNO2

IUPAC Name:

1-chloro-2-nitrobenzene

Details on test material:

- Name of test material (as cited in study report): o-Chlornitrobenzol
- Analytical purity: 99%
- Impurities (identity and concentrations): 1% p-chloronitrobenzene (max.)
- Other: Test compound ordered by Griesheim, Chem. Betriebe 1, Dr. Görtz
- Stability: no data

Method

Target gene:

S. typhimurium: histidin
E. coli: tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat (Aroclor 1254 induced) liver S9-mix

Test concentrations with justification for top dose:

0, 4, 20, 100, 500, 2500 µg/plate, dissolved in 100 µL DMSO, additionally:TA100 with S9-mix: 2000 µg/plate, dissolved in 100 µL DMSO

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

for concentrations see details below

Positive control substance:

other: sodium azide; 9-Aminoacridine; 2 -Nitrofluorene; N-Methyl-N´-nitro-N-nitrosoguanidine; Benzo[a]pyrene; 2 -Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation);

DURATION
- Exposure duration: 48-72 hours

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: other: Surviving fraction; bacterial background lawn

For the plate incorporation method, without metabolic activation, 0.1 ml of the test solutions, 0.1 ml of fresh bacterial culture and 0.5 ml of sterile buffer are mixed with 2.0 ml of overlay agar. For the assay with metabolic activation, usually 0.5 ml of metabolic activation mixture are mixed with the overlay agar (2.0 ml), together with the bacteria and test substance/test solution. All plates were incubated at 37°C for 48-72 hours in the dark. After the incubation period, the number of revertant colonies per plate is counted.

Toxicity and dose finding
Preliminary toxicity tests were performed with all tester strains using a smal number of plates to calculate an appropriate dose range.
In combination, toxicity testing was performed as follows: 0,1 ml of the different solutions of the test compound were mixed with 0.1 ml of 10e-6 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose).

Evaluation criteria:

no data

Statistics:

no data

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium, other: TA 98, TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Test results 5

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 6

Species / strain:

other: TA 1535, TA 1537, WP2uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Data on cytotoxicity were only given for Salmonella typhimurium strain TA 100.
Without metabolic activation no cytotoxicity occured in this strain.
With metabolic activation cytotoxicity (> 50 %) occured at 2500 µg/plate.

No more information given.

Remarks on result:

other: strain/cell type: TA 98

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

TA 100: Number of revertant colonies per plate and mean values of 3 plates             

Dose^1µg/plate	Metabolic activation	Mean value	Colonies per plate	Surviving fraction
0	-	149	169, 134, 143	1,0
4	-	133	160, 130, 110	1,0
20	-	143	148, 143, 138	1,0
100	-	155	174, 164, 128	1,0
500	-	130	172, 91, 127	0,9
1000	-	140	164, 116, 141	0,9
2500	-	21	62, *, * (*)	0,08
0	+	142	136 154, 137	1,0
4	+	135	107, 150, 148	1,1
20	+	137	142, 148, 121	1,1
100	+	148	180, 132, 133	1,1
500	+	249	301, 259, 186	0,9
1000	+	343	460, 380, 190 (*)	0,9
2500	+	170	261, 103, 147 (*)	0,3

Second experiment: TA 100: Number of revertant colonies per plate and mean values

Dose^1µg/plate	Metabolic activation	Mean value	Colonies per plate
0	+	145	141, 151, 150, 136
4	+	140	135, 169, 112, 142
20	+	138	136, 140, 144, 130
100	+	151	154, 149, 150, 150
250	+	175	180, 181, 170, 168
500	+	221	198, 222, 240, 225
1000	+	400	414, 464, 323, 400
2000	+	311	356, 354, 305, 228 (*)

TA 1535: Number of revertant colonies per plate and mean values

Dose^1µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	23	24, 25, 19
4	-	16	17, 12, 19
20	-	23	20, 25, 25
100	-	24	13, 27, 31
500	-	24	34, 19, 19
2500	-	5	0, 8, 8 (*)
0	+	13	15, 10, 13
4	+	10	8, 14, 8
20	+	10	8, 12, 11
100	+	10	10, 6, 13
500	+	11	9, 9, 15
2500	+	9	10, 8, 8 (*)

TA 1537: Number of revertant colonies per plate and mean values

Dose^1µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	5	4, 4, 8
4	-	5	4, 5, 7
20	-	9	12, 5, 9
100	-	9	8, 10, 9
500	-	5	5, 7, 4
2500	-	1	0, 0, 4 (*)
0	+	9	7, 8, 11
4	+	5	3, 6, 6
20	+	7	8, 8, 6
100	+	10	7, 10, 13
500	+	7	6, 4, 11
2500	+	6	10, 2, 5 (*)

TA 1538: Number of revertant colonies per plate and mean values

Dose^1µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	9	5, 13, 9, 8
4	-	9	9, 9, 10, 9
20	-	11	9, 9, 13, 14
100	-	12	6, 12, 12, 16
500	-	16	15, 19, 18, 10
1000	-	22	23, 21, 21, 21
2500	-		16, *, *, * (*)
0	+	17	18, 15, 18, 18
4	+	16	12, 17, 18, 16
20	+	17	13, 18, 21, 15
100	+	16	17, 16, 15, 14
500	+	16	13, 16, 15, 19
1000	+	23	22, 22, 23, 26
2500	+	16	17, 14, 19, 13 (*)

T 98: Number of revertant colonies per plate and mean values

Dose^1µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	22	23, 19, 25
4	-	25	19, 32, 24
20	-	17	19, 19, 13
100	-	21	19, 14, 29
500	-	24	24, 16, 33
1000	-	22	24, 23, 20
2500	-	28	25, 25, 34 (*)
0	+	24	23, 27, 23
4	+	27	20, 32, 29
20	+	27	32, 24, 26
100	+	30	29, 36, 24
500	+	39	37, 37, 42
1000	+	47	36, 48, 57
2500	+	33	36, 36, 27

WP2uvrA: Number of revertant colonies per plate and mean values

Dose^1µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	29	33, 27, 28
4	-	37	34, 38, 39
20	-	36	41, 36, 30
100	-	40	45, 43, 33
500	-	40	43, 39, 37
2500	-	19	20, 14, 22
0	+	37	49, 29 ,32
4	+	35	37, 40, 29
20	+	35	48, 34, 24
100	+	39	42, 29, 47
500	+	44	39, 51, 42
2500	+	42	43, 52, 32

¹: dissolved in DMSO

- : absense

+ : presence

* : incomplete bacterial lawn

Mutability (positive controls) and sterility test of the experiment with o-Chlornitrobenzol

Number of revertant colonies per plate (mean values) using Salmonella typhimurium strains and E. coli

Strain	Compound	Dose (µg/plate)	Metab. activ.	Mean value
TA 100 TA 1535 TA 1537 TA 1538 TA 98 WP2uvrA -	Sodium azide Sodium azide 9-Aminoacridine 2-Nitrofluorene 2-Nitrofluorene MNNG o-Chlornitrobenzol	1 1 50 5 5 2.5 500	- - - - - - -	726 481 202 843 727 376 0
TA 100 TA 100^2 TA 1535 TA 1537 TA 1538 TA 98 WP2uvrA	2-Aminoanthracene 2-Aminoanthracene 2-Aminoanthracene 2-Aminoanthracene 2-Aminoanthracene 2-Aminoanthracene 2-Aminoanthracene	0.5 0.5 1 1 0.5 0.5 10	+ + + + + + +	649 536 289 222 546 956 830
TA 100 TA 100 TA 1535 TA 1537 TA 1538 TA 98 WP2uvrA - -	Benzo[a]pyrene Benzo[a]pyrene Benzo[a]pyrene Benzo[a]pyrene Benzo[a]pyrene Benzo[a]pyrene Benzo[a]pyrene S9 mix o-Chlornitrobenzol	10 10 10 10 10 10 10 500 µl 500 µg	+ + + + + + + + +	643 619 26 152 219 720 124 0 0

- : absence

+ : presence

² : second experiment

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation in TA 100
ambiguous with metabolic activation TA 98
ambiguous without metabolic activation in TA 1538
negative in TA 1537, TA 1535 and ind WP2uvrA

Executive summary:

Hoechst AG, 1984

The mutagenic activity of 1 -chloro-2-nitrobenzene was investigated in a bacterial gene mutation assay conducted similar to OECD-guideline 471. The preincubation assay was performed with Salmonella typhimurium TA98, TA1538, TA1537, TA100, TA1535 and in E. coli strain WP2uvrA both in presence and absence of a metabolic activator (Aroclor1254-induced rat liver S9) at 0, 4, 20, 100, 500, 2500 µg and additionally in TA100 with S9 -Mix 2000 µg 1 -chloro-2 -nitrobenzene per plate.

In the absence of the metabolic activation system, the test compound induced a relatively small but dose dependent increase of revertants with the bacterial strain TA1538 (2.4 times the control value). 1 -chloro-2 -nitrobenzene induced mutations in presence of the metabolic activator in the strain TA100.