3,3'-(1,4-phenylenediimino)bis[4,5,6,7-tetrachloro-1H-isoindol-1-one]

Genetic toxicity: in vitro

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

Test material information

Constituent 1

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.