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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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
Justification for type of information:
The study contains experimental data of the registered substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21July 1997, corrected 26 June 2020
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3'-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]
EC Number:
226-999-5
EC Name:
3,3'-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]
Cas Number:
5590-18-1
Molecular formula:
C22H6Cl8N4O2
IUPAC Name:
3,3'-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]
Test material form:
solid: particulate/powder
Remarks:
Yellow powder
Details on test material:
- Name of test material (as cited in study report): 3,3'-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]
- Molecular formula (if other than submission substance): C22H6Cl8N4O2
- Molecular weight (if other than submission substance): 641.939 g/mol
- Smiles notation (if other than submission substance): C=1(c2c(c(c(Cl)c(Cl)c2C(=O)N1)Cl)Cl)Nc1ccc(cc1)NC1=NC(c2c1c(c(c(c2Cl)Cl)Cl)Cl)=O
- InChl (if other than submission substance): 1S/C22H6Cl8N4O2/c23-11-7-9(13(25)17(29)15(11)27)21(35)33-19(7)31-5-1-2-6(4-3-5)32-20-8-10(22(36)34-20)14(26)18(30)16(28)12(8)24/h1-4H,(H,31,33,35)(H,32,34,36)
- Substance type: Organic
- Physical state: Yellow powder
- Physical state: 99%
Specific details on test material used for the study:
Purity:99%

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9 metabolic activation system was used. An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to get a final concentration of approximately 10 % v/v in the S9 mix
Composition of S9 mix:
D-glucose-6-phosphate 0.8 g
β-NADP 1.75 g
MgCl2 1.0 g
KCl 1.35 g
Na2HPO4.H2O 6.4 g
NaH2PO4.H2O 1.4 g
Test concentrations with justification for top dose:
0.0 (NC), 0.0 (VC), 0.156, 0.313, 0.625, 1.250 and 2.50 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was found insoluble in distilled water. Therefore, the solubility was checked in Dimethyl Sulphoxide (DMSO) and found soluble at 50 mg/mL. Thus, DMSO was chosen as a solvent for pre-experiment, Trial I and Trial II.
Controls
Untreated negative controls:
yes
Remarks:
Distilled water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)
Details on test system and experimental conditions:
For each strain and dose level, including controls, three plates (triplicate) were used. For the plate incorporation method (Trial I), the following materials were mixed in a test tube and poured onto the selective agar plates:
100 µL Test solution at each dose level, negative control, vehicle control, and reference mutagen solution (positive control),
500 µL S9 mix (for the test with metabolic activation) or S9 mix substitution buffer (for the test without metabolic activation),
100 µL Bacterial suspension,
2000 µL Overlay agar
For the preincubation assay (Trial II), 100 µL test solution, 500 µL S9 mix and S9 mix substitution buffer and 100 µL bacterial suspensions were mixed in a test tube and incubated at 37 °C for 60 minutes. After preincubation, 2.0 mL overlay agar (47°C) was added to each tube. The mixture was poured on minimal agar plates. After solidification, the plates were incubated in an inverted position for 48 hours at 37 °C.
Evaluation criteria:
A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.
A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration was judged as biologically relevant if it was reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative control and vehicle control, the increase was not considered biologically relevant.

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Results of the solubility test:
The test item was found insoluble in distilled water. Therefore, the solubility was checked in Dimethyl Sulphoxide (DMSO) and found soluble at 50 mg/mL. Thus, DMSO was chosen as a solvent for pre-experiment, Trial I and Trial II.
Results of the precipitation test:
Insolubility was assessed as precipitation in the final mixture under the actual test conditions and evident to the unaided eye. The test item was dissolved in DMSO at 50 mg/mL concentration and checked for precipitation on agar. Precipitation was observed at 5 and 3.5 mg/plate. Slight precipitation was noted at 2.5 mg/plate.
The test item was further assessed for precipitation at 3.5, 3.0 and 2.75 mg/plate concentrations. The results are the follows:
Precipitation was observed at 3.5 mg/plate, 3.0 mg/plate and 2.75 mg/plate concentrations, which was considered to interfere with the colony count. Therefore 2.5 mg/plate was selected as the highest concentration for pre-experiment.
Preliminary cytotoxicity test:
The cytotoxicity test was performed with strains TA98 and TA100. Eight concentrations (i.e. 0.0 (negative control; NC), 0.0 (vehicle control; VC), 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate) with half-log intervals (√10) were tested for toxicity and mutation induction with 3 plates each (triplicates). In TA98 and TA100 tester strains, no reduction in revertant colonies but moderate inhibition of background lawn was observed at 2.5 mg/plate (T8) both in the presence and absence of S9 metabolic activation. No reduction in colony count and diminished background lawn was observed at concentrations of 0.791 – 0.001 mg/plate (T7-T1) neither in the presence (+S9) nor the absence (-S9) of metabolic activation. Based on the results of the pre-experiment, the following doses were selected for the main study trials:0.0 (NC), 0.0(VC), 0.156, 0.313, 0.625, 1.250 and 2.5 mg/plate, both in the absence (-S9) and presence of metabolic activation (+S9).

Remarks on result:
other: No mutagenic potetial

Any other information on results incl. tables

ABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)

R

Absence of Metabolic Activation

 (-S9)

Presence of Metabolic Activation (+S9)

TA100

TA 98

TA100

TA 98

NC

(0.00)

R1

114

20

116

19

R2

102

18

120

24

R3

116

21

110

22

VC

(0.00)

R1

110

24

128

25

R2

126

20

124

24

R3

118

26

114

28

T1

(0.001)

R1

110

25

120

23

R2

124

20

124

26

R3

118

23

116

24

T2

(0.003)

R1

122

22

118

25

R2

110

20

114

20

R3

116

23

120

23

T3

(0.008)

R1

120

23

124

25

R2

108

21

108

23

R3

114

18

112

26

T4

(0.025)

R1

114

21

112

25

R2

124

24

122

24

R3

112

22

120

26

T5

(0.079)

R1

114

19

118

21

R2

112

22

120

25

R3

118

20

112

24

T6

(0.250)

R1

110

22

114

22

R2

114

23

110

21

R3

116

21

122

24

T7

(0.791)

R1

110

19

116

26

R2

116

20

112

23

R3

120

21

120

23

T8

(2.5)

R1

104 (+++)

21 (+++)

118 (+++)

23 (+++)

R2

118 (+++)

23 (+++)

110 (+++)

22 (+++)

R3

112 (+++)

20 (+++)

114 (+++)

26 (+++)

PC

R1

1192

904

1184

1208

R2

1232

848

1328

1280

R3

1056

888

1200

1152

NC          =     Negative control; VC   =          Vehicle Control; PC            =            Positive control

R             =     Replicate

T             =     Test concentration (T8: Highest, T1: Lowest)

Background Lawn: +++ = Moderate Inhibition

4-Nitro-o-phenylenediamine [10μg/plate]: TA 98

Sodium azide [10μg/plate]: TA 100,

2-Aminoanthracene [2.5μg/plate]: TA 98, TA 100

TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD
TRIAL I

Dose (mg/plate)

R

Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

5

12

24

118

244

R2

3

11

20

112

228

R3

4

13

22

106

240

VC.

(0.00)

R1

7

15

25

120

244

R2

5

13

24

126

260

R3

6

17

26

118

252

T1

(0.156)

R1

4

15

23

118

252

R2

7

13

25

114

240

R3

6

14

24

120

248

T2

(0.313)

R1

5

11

22

112

240

R2

4

15

24

118

248

R3

7

14

22

114

232

T3

(0.625)

R1

4

13

21

120

248

R2

5

16

22

112

236

R3

5

14

23

116

240

T4

(1.250)

R1

4

13

23

110

244

R2

5

12

24

116

236

R3

4

14

23

120

248

T5

(2.5)

R1

5

14

21

114

240

R2

5

12

25

112

248

R3

5

14

21

116

228

PC

R1

168

456

1188

1308

1428

R2

200

336

1224

1164

1464

R3

188

384

1140

1212

1368

 

Dose (mg/plate)

R

Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

3

10

19

110

224

R2

3

13

21

104

240

R3

4

11

18

114

232

VC.

(0.00)

R1

5

14

23

124

236

R2

6

16

22

120

256

R3

5

12

25

116

248

T1

(0.156)

R1

4

13

22

114

232

R2

4

11

24

120

252

R3

6

14

20

112

240

T2

(0.313)

R1

6

15

21

108

228

R2

5

10

23

114

244

R3

6

14

20

112

236

T3

(0.625)

R1

5

12

23

114

236

R2

6

15

20

110

252

R3

4

13

24

118

240

T4

(1.250)

R1

4

14

21

110

248

R2

4

10

20

112

232

R3

4

11

19

116

240

T5

(2.5)

R1

4

12

20

110

228

R2

5

14

21

116

248

R3

4

11

22

114

236

PC

R1

172

1272

848

1236

1440

R2

164

1188

1008

1152

1608

R3

192

1224

896

1176

1536

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                                                        2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100,  2- Aminoanthracene [10μg/plate]:TA 102,                                                      Sodium azide [10μg/plate]: TA 1535, TA 100, 

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate],    Methyl methanesulfonate [4μl/plate]: TA 102.

TABLE 3 - REVERTANT COUNT IN PREINCUBATION METHOD TRIAL II

Dose (mg/plate)

R

In the Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

5

14

22

108

236

R2

4

11

21

114

244

R3

4

14

20

110

240

VC.

(0.00)

R1

6

16

25

118

256

R2

7

17

27

124

272

R3

6

15

26

116

268

T1

(0.156)

R1

5

14

23

110

244

R2

7

16

25

116

256

R3

6

13

22

114

240

T2

(0.313)

R1

7

15

25

118

232

R2

4

12

22

112

248

R3

5

14

24

114

252

T3

(0.625)

R1

5

16

22

110

244

R2

5

14

21

116

240

R3

5

15

23

112

236

T4

(1.250)

R1

4

14

22

118

252

R2

7

13

21

110

232

R3

6

15

25

114

244

T5

(2.5)

R1

5

16

21

108

248

R2

6

13

24

116

240

R3

4

15

25

112

236

PC

R1

168

376

972

1368

1380

R2

144

320

1212

1404

1272

R3

160

416

1152

1320

1320

 

 

Dose

(mg/plate)

R

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

3

11

22

110

232

R2

4

10

18

104

228

R3

3

12

21

112

236

VC.

(0.00)

R1

5

15

25

120

242

R2

7

13

23

114

260

R3

6

16

24

118

256

T1

(0.156)

R1

5

13

21

106

236

R2

6

15

26

118

252

R3

4

14

22

112

244

T2

(0.313)

R1

6

15

25

114

248

R2

5

11

23

116

236

R3

6

12

20

108

240

T3

(0.625)

R1

5

12

24

118

240

R2

4

15

22

112

232

R3

4

13

21

110

244

T4

(1.250)

R1

5

14

24

108

240

R2

5

12

21

106

232

R3

6

13

20

114

236

T5

(2.5)

R1

6

14

21

114

236

R2

4

11

23

106

244

R3

4

12

22

112

240

PC

R1

152

840

612

1140

1416

R2

176

1104

828

1332

1344

R3

168

924

720

1284

1512

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                                                         2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100,        
2-Aminoanthracene [10μg/plate]:TA 102,                                                        Sodium azide [10μg/plate]: TA 1535, TA 100,                                                                                                                 

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate],       Methyl methanesulfonate [4μl/plate]: TA 102.

 

TABLE 4 - MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD TRIALI

Dose (mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

4.00

1.00

12.00

1.00

22.00

2.00

112.00

6.00

237.33

8.33

VC.

(0.00)

6.00

1.00

15.00

2.00

25.00

1.00

121.33

4.16

252.00

8.00

T1

(0.156)

5.67

1.53

14.00

1.00

24.00

1.00

117.33

3.06

246.67

6.11

T2

(0.313)

5.33

1.53

13.33

2.08

22.67

1.15

114.67

3.06

240.00

8.00

T3

(0.635)

4.67

0.58

14.33

1.53

22.00

1.00

116.00

4.00

241.33

6.11

T4

(1.250)

4.33

0.58

13.00

1.00

23.33

0.58

115.33

5.03

242.67

6.11

T5

(2.5)

5.00

0.00

13.33

1.15

22.33

2.31

114.00

2.00

238.67

10.07

PC

185.33

16.17

392.00

60.40

1184.00

42.14

1228.00

73.32

1420.00

48.50

 

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

3.33

0.58

11.33

1.53

19.33

1.53

109.33

5.03

232.00

8.00

VC

(0.00)

5.33

0.58

14.00

2.00

23.33

1.53

120.00

4.00

246.67

10.07

T1

(0.156)

4.67

1.15

12.67

1.53

22.00

2.00

115.33

4.16

241.33

10.07

T2

(0.313)

5.67

0.58

13.00

2.65

21.33

1.53

111.33

3.06

236.00

8.00

T3

(0.635)

5.00

1.00

13.33

1.53

22.33

2.08

114.00

4.00

242.67

8.33

T4

(1.250)

4.00

0.00

11.67

2.08

20.00

1.00

112.67

3.06

240.00

8.00

T5

(2.5)

4.33

0.58

12.33

1.53

21.00

1.00

113.33

3.06

237.33

10.07

PC

176.00

14.42

1228.00

42.14

917.33

82.11

1188.00

43.27

1528.00

84.29

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                       

Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                  

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

 

TABLE 5 - MEAN REVERTANT COUNT IN PREINCUBATIONMETHOD
TRIAL II

Dose

(mg/plate)

Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

4.33

0.58

13.00

1.73

21.00

1.00

110.67

3.06

240.00

4.00

VC

(0.00)

6.33

0.58

16.00

1.00

26.00

1.00

119.33

4.16

265.33

8.33

T1

(0.156)

6.00

1.00

14.33

1.53

23.33

1.53

113.33

3.06

246.67

8.33

T2

(0.313)

5.33

1.53

13.67

1.53

23.67

1.53

114.67

3.06

244.00

10.58

T3

(0.635)

5.00

0.00

15.00

1.00

22.00

1.00

112.67

3.06

240.00

4.00

T4

(1.250)

5.67

1.53

14.00

1.00

22.67

2.08

114.00

4.00

242.67

10.07

T5

(2.5)

5.00

1.00

14.67

1.53

23.33

2.08

112.00

4.00

241.33

6.11

PC

157.33

12.22

370.67

48.22

1112.00

124.90

1364.00

42.14

1324.00

54.11

 

Dose

(mg/plate)

Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

3.33

0.58

11.00

1.00

20.33

2.08

108.67

4.16

232.00

4.00

VC

(0.00)

6.00

1.00

14.67

1.53

24.00

1.00

117.33

3.06

252.67

9.45

T1

(0.156)

5.00

1.00

14.00

1.00

23.00

2.65

112.00

6.00

244.00

8.00

T2

(0.313)

5.67

0.58

12.67

2.08

22.67

2.52

112.67

4.16

241.33

6.11

T3

(0.635)

4.33

0.58

13.33

1.53

22.33

1.53

113.33

4.16

238.67

6.11

T4

(1.250)

5.33

0.58

13.00

1.00

21.67

2.08

109.33

4.16

236.00

4.00

T5

(2.5)

4.67

1.15

12.33

1.53

22.00

1.00

110.67

4.16

240.00

4.00

PC

165.33

12.22

956.00

134.88

720.00

108.00

1252.00

99.92

1424.00

84.29

NC= Negative Control,VC= Vehicle Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

2-Aminoanthracene [10μg/plate]: TA 102

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537 [50μg/plate] TA 98 [10μg/plate]

Methyl methanesulfonate: [4μl/plate]: TA 102

 

 HISTORICAL CONTROL DATA

These data represent the laboratory's historical control data.

Trial I (Plate Incorporation Method)

Strains

Metabolic Activation

Treatment

Mean

SD

Max

Min

TA 1537

S9 +

Negative control

6

2

10

2

S9 -

6

2

10

2

S9 +

Solvent control

6

2

10

2

S9 -

6

2

10

2

S9 +

Positive control

168

38

245

92

S9 -

175

43

261

89

TA 1535

S9 +

Negative control

12

3

18

7

S9 -

12

3

18

7

S9 +

Solvent control

13

3

18

7

S9 -

13

3

18

7

S9 +

Positive control

336

211

757

86

S9 -

1200

263

1726

674

TA 98

S9 +

Negative control

24

6

36

11

S9 -

23

6

35

11

S9 +

Solvent control

25

6

37

13

S9 -

23

5

33

13

S9 +

Positive control

1099

312

1722

476

S9 -

815

284

1383

248

TA 100

S9 +

Negative control

117

28

173

61

S9 -

114

26

166

62

S9 +

Solvent control

116

28

172

60

S9 -

113

26

165

61

S9 +

Positive control

1488

390

2268

709

S9 -

1311

298

1906

715

TA 102

S9 +

Negative control

274

42

358

190

S9 -

271

55

382

161

S9 +

Solvent control

279

65

409

150

S9 -

277

82

442

112

S9 +

Positive control

1648

305

2258

1037

S9 -

1896

364

2624

1168

Mean = mean value of revertants/plate, SD = standard deviation, Min = -2SD, Max = +2SD

 

 


HISTORICAL CONTROL DATA (Contd.)

Trial II (Pre-Incubation Method)

Strains

Metabolic Activation

Treatment

Mean

SD

Max

Min

TA 1537

S9 +

Negative control

6

2

10

2

S9 -

6

2

10

2

S9 +

Solvent control

6

2

10

3

S9 -

6

2

10

2

S9 +

Positive control

170

39

249

91

S9 -

182

43

268

96

TA 1535

S9 +

Negative control

13

3

18

7

S9 -

12

3

18

7

S9 +

Solvent control

13

3

18

8

S9 -

13

3

18

7

S9 +

Positive control

299

197

694

145

S9 -

1244

260

1765

724

TA 98

S9 +

Negative control

24

6

35

13

S9 -

23

5

33

13

S9 +

Solvent control

24

5

35

14

S9 -

23

5

32

14

S9 +

Positive control

1269

275

1819

719

S9 -

740

210

1160

320

TA 100

S9 +

Negative control

117

25

166

67

S9 -

113

23

159

66

S9 +

Solvent control

116

22

159

73

S9 -

112

20

151

73

S9 +

Positive control

1469

347

2163

775

S9 -

1352

263

1878

827

TA 102

S9 +

Negative control

281

32

345

218

S9 -

276

28

331

220

S9 +

Solvent control

281

34

350

212

S9 -

276

34

344

207

S9 +

Positive control

1595

287

2168

1022

S9 -

1753

248

2248

1258

Mean = mean value of revertants/plate, SD = standard deviation, Min = -2SD, Max = +2SD

Applicant's summary and conclusion

Conclusions:
The registered chemical 3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one] (CAS No. 5590-18-1) did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system in study performed according to OECD TG 471.
Executive summary:

A bacterial reverse mutation assay (OECD TG 471) was performed to investigate the potential of the registered chemical 3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one] (CAS No. 5590-18-1) to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments with and without S9 metabolic activation system. The test concentrations were selected based on the results of a pre-liminary cytotoxicity test. The mutagenicity test was performed with the following test substance concentrations: 0 (NC), 0 (VC),  0.156, 0.313, 0.625, 1.250 and 2.5 mg/plate in both trials and in the presence (+S9) and absence of metabolic activation (-S9). Results: No significant increase in the number of revertant colonies, either in the presence or absence of metabolic activation was observed at any concentrations tested as compared to the vehicle control. No trend of an increased number of revertant colonies with increased dosing of the test item was observed. The spontaneous reversion rates in the negative and vehicle controls were within the historical range. Each strain-specific positive control in Trial I and II showed a significant increase in the number of revertant colonies. Conclusion:The registered chemical 3,3’-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one] (CAS No. 5590-18-1) did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation in study performed according to OECD TG 471.