Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1979
Report Date:
1979

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Not to GLP
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
2,4-TDA CAS 95-80-7
2,6-TDA CAS 823-40-5

Method

Species / strain
Species / strain / cell type:
other: Salmonella typhimurium strains, TA100, TA98, TA1535, TA1537 and TA1538
Metabolic activation:
with
Metabolic activation system:
Aroclor 1254 induced rat liver S-9
Vehicle / solvent:
dimethylsulphoxide (DMSO)

Results and discussion

Additional information on results:
2,4-Toluenediamine (2,4-TDA) was positive in the presence of S-9 mix in Salmonella typhimurium strains TA 98, TA 100, TA 1537 and TA 1538 with respect to induction of bacterial gene mutations.

2,4-Toluenediamine (2,4-TDA) was negative in the absence of S-9 mix in all Salmonella typhimurium strains tested, with respect to induction of bacterial gene mutations.

2,6-Toluenediamine (2,6-TDA) was positive in the presence of S-9 mix in Salmonella typhimurium strains TA 98, TA 100, TA 1537 and TA 1538 with respect to induction of bacterial gene mutations.

2,6-Toluenediamine (2,6-TDA) was negative in the absence of S-9 mix in all Salmonella typhimurium strains tested, with respect to induction of bacterial gene mutations.

See Tables 1 and 2. Positive controls were used as detailed in Tables 1 and 2 (see Remarks on Results below). Toxicity data are available in Table 3 (see Remarks on Results below).

Any other information on results incl. tables

Table 1. Bacterial mutagenicity of 2,4-TDA (see Additional information on results, above)

Chemical

Amount per plate (ug)

S-9 (b)

Revertants per plate (a)

TA98

TA100

TA1535

TA1537

TA1538

Negative Control

-

-

13

54

54

6

8

Negative Control

-

+

17

55

52

7

11

Positive Control (c)

-

-

558

237

78

122

1209

Positive Control (d)

-

+

1178

242

0

379

1156

2,4-TDA

500

-

14

67

61

9

10

1077

-

12

76

60

8

6

2320

-

13

80

63

8

12

5000

-

10

72

58

8

11

500

+

213

98

46

23

154

1077

+

376

140

56

34

362

2320

+

738

167

66

82

750

5000

+

1144

218

60

101

1138

(a) Average of two plates <br>(b) Aroclor 1254 induced Fisher 344 rat liver.<br>(c) The following positive controls were used without activating medium: dinitrotoluene (500 ug, TA1535 and TA100), quinacrine mustard (10 ug, TA1537) and nitrofluorene (100 ug, TA1538 and TA98).<br>(d) The following positive controls were used with activating medium: anthramine (100 ug, TA1535 and TA 100), aminoquinoline (100 ug, TA1537) and acetamidofluorene (500 ug, TA1538 and TA98).

Table 2. Bacterial mutagenicity of 2,6-TDA (see Additional information on results, above)

Chemical

Amount per plate (ug)

S-9 (b)

Revertants per plate (a)

TA98

TA100

TA1535

TA1537

TA1538

Negative Control

-

-

13

54

54

6

8

Negative Control

-

+

17

55

52

7

11

Positive Control (c)

-

-

558

237

78

122

1209

Positive Control (d)

-

+

1178

242

0

379

1156

2,6-TDA

500

-

12

72

64

10

7

1077

-

14

64

63

6

8

2320

-

14

80

62

8

14

5000

-

12

74

66

6

10

500

+

151

418

66

20

176

1077

+

205

645

61

20

261

2320

+

348

1086

66

30

380

5000

+

395

1000

64

34

528

(a) Average of two plates. <br>(b) Aroclor 1254 induced Fisher 344 rat liver.<br>(c) The following positive controls were used without activating medium: dinitrotoluene (500 ug, TA1535 and TA100), quinacrine mustard (10 ug, TA1537) and nitrofluorene (100 ug, TA1538 and TA98).<br>(d) The following positive controls were used with activating medium: anthramine (100 ug, TA1535 and TA 100), aminoquinoline (100 ug, TA1537) and acetamidofluorene (500 ug, TA1538 and TA98).

Table 3. Toxicity of TDA isomers for S. Typhimurium (see Additional information on results, above)

Chemical

Amount per plate (ug)

S-9 (b)

Relative survival (a)

TA98

TA100

TA1535

TA1537

TA1538

2,4-TDA

500

-

0.150

0.776

0.776

1.032

0.453

2,4-TDA

1077

-

0.183

0.680

0.629

0.886

0.432

2,4-TDA

2320

-

0.187

0.482

0.755

0.889

0.456

2,4-TDA

5000

-

0.162

-

0.598

0.912

0.345

2,4-TDA

500

+

0.406

0.541

1.083

1.054

0.506

2,4-TDA

1077

+

0.240

0.646

0.882

0.931

0.424

2,4-TDA

2320

+

0.233

0.584

0.802

0.915

0.367

2,4-TDA

5000

+

0.229

0.619

0.917

0.905

0.357

2,5-TDA

500

-

0.000

0.000

0.595

0.076

0.042

2,5-TDA

1077

-

0.000

0.000

0.269

0.0024

0.000

2,5-TDA

2320

-

0.000

0.000

0.000

0.000

0.000

2,5-TDA

5000

-

0.000

0.000

0.000

0.000

0.000

2,5-TDA

500

+

0.222

0.578

1.051

0.860

0.287

2,5-TDA

1077

+

0.257

0.438

0.962

0.860

0.284

2,5-TDA

2320

+

0.017

0.000

0.683

0.231

0.209

2,5-TDA

5000

+

0.000

0.000

0.000

0.000

0.000

2,6-TDA

500

-

0.150

0.488

0.898

0.927

0.356

2,6-TDA

1077

-

0.158

0.540

1.033

0.931

0.355

2,6-TDA

2320

-

0.166

0.475

0.771

0.907

0.335

2,6-TDA

5000

-

0.168

0.409

0.733

0.912

0.342

2,6-TDA

500

+

0.208

0.511

0.993

1.015

0.295

2,6-TDA

1077

+

0.205

0.374

0.948

0.856

0.302

2,6-TDA

2320

+

0.200

0.473

1.078

0.808

0.334

2,6-TDA

5000

+

0.259

0.464

0.990

0.927

0.332

3,4-TDA

1000

-

0.107

0.463

0.803

0.794

0.341

3,4-TDA

1710

-

0.094

0.473

0.767

0.811

0.302

3,4-TDA

2924

-

0.068

0.471

0.598

0.712

0.185

3,4-TDA

5000

-

0.018

0.411

0.407

0.326

0.042

3,4-TDA

1000

+

0.224

0.471

0.901

0.872

0.320

3,4-TDA

1710

+

0.247

0.501

0.904

0.868

0.313

3,4-TDA

2924

+

0.240

0.422

0.816

0.861

0.309

3,4-TDA

5000

+

0.194

0.385

0.773

0.875

0.309

(a) The amount of chemical indicated, + or - rat S-9, was added to a tube of top agar containing 0.1 ml of a 10-6 dilution of the relevant tester strain. The mixture then was poured onto a nutrient agar plate, was allowed to harden, and the plates were incubated at 37°C for 24 hr. <br>Relative survival = <br>Number of colonies on plate + chemical <br>Number of colonies on control plate <br>(b) Aroclor 1254 induced Fisher 344 rat liver

There are various other in vivo bacterial mutation assays reported on TDA isomers. The EU Risk Assessment for 2,4-TDA discusses these reports thus:

"Bacterial gene mutations

Bacterial genotoxicity tests were clearly positive with S-9 mix in several tests. 2,4-Toluenediamine (2,4-TDA) was positive with S-9 mix in Salmonella typhimurium strains TA 98, TA 100, TA 1537 and TA 1538 with respect to induction of bacterial gene mutations for doses from 20 µg/plate upward (JETOC, 1996; George and Westmoreland, 1991; Shahin et al., 1985; Haworth et al., 1983; Shahin et al., 1980; Green et al, 1979). All investigations showed strong and dose-dependent effects; no toxic effects were observed. These Salmonella typhimurium strains were negative without S-9 mix up to dose of 10000 µg/plate. 2,4-TDA was also positive in TA98 with S-9 mix in a microsuspension bioassay (George et al., 2001).

Cunningham and Matthews (1990) described the role of bacterial acetyltransferase on mutagenic effect of 2,4-TDA in bacterial mutation test with metabolic activation as shown by experiments with strain Salmonella typhimurium strain TA98/1,8-DNP6 (deficient in acetyltransferase), TA 98 (normal acetyltransferase) and the overproducer of acetyltransferase TA 98(pYG219). Compared with TA 98 the effect of strain TA98/1,8-DNP6 resulted in approximately 90% decrease in the mutagenic potency whereas the strain TA 98(pYG219) greatly enhanced the mutagenic effect. The authors concluded that after N-hydroxylation of 2,4-TDA by S-9 mix cytochrome P450 the resulting hydroxylamino intermediate is further activated by bacterial acetyltransferase to form the ultimate reactive intermediate, which is postulated to be 4-acetoxyamino-2-aminotoluene.

Negative results with and without S-9 mix were obtained in Salmonella typhimurium strain TA 1535 up to 10000 µg/plate (JETOC, 1996; Haworth et al., 1983) and in E. coli WP2uvrA up to 5000 µg/plate (JETOC, 1996)."

Table 1.   In vitro tests: bacterial genotoxicity

Test system

Concentration range

Result

Toxicity

Remarks

Reference

with S-9 mix

without S-9 mix

Gene mutation, Salm. typh. TA 98, TA 100, TA 1535, TA 1537, E.coli WP2uvrA

0.07 63- 5000

µg/plate

0.07 63- 5000

µg/plate

positive

no toxic effects

positive only with S-9 mix

JETOC, 1996

Gene mutation, Salm. typh. TA 98

100 - 3333 µg/plate

not done

positive

no toxic effects

 .

George and Westmoreland, 1991

Gene mutation, Salm. typh. TA 97, TA 1537, TA 1538

50 - 500 µg/plate

not done

positive

no toxic effects

 .

Shahin et al., 1985

Gene mutation, Salm. typh. TA 98, TA 100, TA 1535, TA 1537

10 - 10'000 µg/plate

10 - 10'000 µg/plate

positive

no toxic effects

positive only with S-9 mix

Haworth et al., 1983

Gene mutation, Salm. typh. TA 98, TA 100, TA 1538

5.0 -1000 µg/plate

5.0 - 1000 µg/plate

positive

no toxic effects

positive only with S-9 mix

Shahin et al., 1980

Gene mutations Salm. typh. TA 98; TA 100, TA 1535, TA 1537, TA 1538

500 - 5000 µg/plate

500 - 5000 µg/plate

positive

no toxic effects

positive only with S-9 mix

Green et al., 1979

Gene mutation, Salm. typh. TA 98

100 - 1666 µg/plate

not done

positive

no data

.

Cunningham and Matthews, 1990

Gene mutation, Salm. typh. TA 98, TA 100 (microsuspension bioassay)

10-500 µg/plate

10-500 µg/plate

positive

no data

positive only with S-9 mix

George et al., 2001

These studies identified in the EU Risk Assessment are consistent with the results of the industry Key endpoint study.

Applicant's summary and conclusion

Conclusions:
Both 2,4-TDA and 2,6-TDA were capable of producing frameshift and basepair substitution mutations in S. typhimurium, after metabolic activation by induced rat liver S-9 fraction.