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Toxicological information

Genetic toxicity: in vitro

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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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 / strainopen allclose all
Species / strain:
other: TA 98, TA 100, TA 1535, TA 1537, TA 1538
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat (Aroclor 1254 induced) liver S9-mix
Species / strain:
E. coli WP2 uvr A
Additional strain characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat (Aroclor 1254 induced) 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:
DMSO
Controls
Negative controls:
no
Solvent 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 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 resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA 98
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
At 2500 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA 100
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity:
yes
Remarks:
At 2500 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Species / strain:
other: TA 98, TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
At 2500 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
not specified
Remarks on result:
other: strain/cell type: TA 98, TA 100
Remarks:
Migrated from field 'Test system'.
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
At 2500 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA 1538
Remarks:
Migrated from field 'Test system'.
Species / strain:
other: TA 1535, TA 1537, WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
At 2500 µg/plate
Vehicle controls valid:
yes
Negative controls valid:
not specified
Positive controls valid:
yes
Remarks on result:
other: strain/cell type: TA 1535, TA 1537, WP2uvrA
Remarks:
Migrated from field 'Test system'.
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.

Any other information on results incl. tables

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

Dose1µg/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

Dose1µg/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

Dose1µg/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

Dose1µg/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

Dose1µg/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

Dose1µg/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

Dose1µg/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 1002

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.