Reactions mass of N-{2-[(4-bromo-6-substituted-2-alkyl-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl)diazenyl]-[alkyl(2-methoxyalkyl)amino]phenyl}acetamide) and N-{2-[(4-bromo-6-substiuted-2-alkyl-1,3-dioxo-2,3-dihydro-1H-isoindol-5-yl)diazenyl]-[alkyl(2-hydroxy-3-phenoxypropyl)amino]phenyl}acetamide

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:

19 February 2014 to 26 March 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

Species / strain

Species / strain / cell type:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/ß-Naphthoflavone induced rat liver S9 (experiment I) and non-induced hamster liver S9 (experiment II)

Test concentrations with justification for top dose:

3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / pre-experiment/experiment I
10; 33; 100; 333; 1000; 2500; and 5000 µg/plate / experiment II

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: suspended in DMSO
- Justification for choice of solvent/vehicle: best suitable solvent

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate incorporation; preincubation;


DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours


NUMBER OF REPLICATIONS: 3 plates


DETERMINATION OF CYTOTOXICITY
A reduction in the number of spontaneous revertants (below the induction factor of 0.5) or a clearing of the bacterial background lawn.

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 WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Species / strain:

other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

in strains TA 1535 and TA 1537

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS

- Water solubility: not soluble
- Precipitation:
The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate in experiment I and from 1000 to 5000 µg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.
- Other confounding effects: none
COMPARISON WITH HISTORICAL CONTROL DATA: performed no deviation
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred most of the strains. A minor reduction in the number of revertants (below the indication factor of 0.5) was observed in strains TA 1535 and TA 1537 in experiment I without S9 mix at 2500 µg/plate and in experiment II in strain TA 1537 in the absence of metabolic activation at 5000 µg/plate and in strain TA 1535 in the presence of metabolic activation at 5000 µg/plate.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table1     Summary of Experiment I

Study Name: 1610101	Study Code: Harlan CCR 1610101
Experiment: 1610101 VV Plate	Date Plated: 19/02/2014
Assay Conditions:	Date Counted: 26/02/2014

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			18 ± 1	14 ± 0	21 ± 4	83 ± 10	47 ± 8
	Untreated			18 ± 4	10 ± 4	26 ± 5	97 ± 6	48 ± 11
	FAT 41044/A	3 µg		17 ± 4	12 ± 2	27 ± 4	86 ± 8	48 ± 6
	TE	10 µg		14 ± 0	13 ± 4	21 ± 4	89 ± 5	53 ± 5
		33 µg		14 ± 1	10 ± 1	19 ± 3	84 ± 9	45 ± 5
		100 µg		18 ± 4	10 ± 2	25 ± 5	97 ± 12	43 ± 10
		333 µg		16 ± 3^P	9 ± 1^P	21 ± 2^P	92 ± 11^P	43 ± 1^P
		1000 µg		10 ± 2^P	8 ± 2^P	15 ± 4^P	91 ± 10^P	44 ± 3^P
		2500 µg		8 ± 1^P	6 ± 3^P	20 ± 4^P	91 ± 10^P	38 ± 4^P
		5000 µg		10 ± 1^P M	6 ± 2^P M	17 ± 1^P M	81 ± 6^P M	37 ± 2^P M
	NaN3	10 µg		2630 ± 130			2117 ± 90
	4-NOPD	10 µg				287 ± 30
	4-NOPD	50 µg			59 ± 2
	MMS	2.0 µL						856 ± 40
With Activation	DMSO			13 ± 4	14 ± 2	29 ± 2	111 ± 8	55 ± 9
	Untreated			18 ± 6	14 ± 2	42 ± 8	126 ± 6	62 ± 4
	FAT 41044/A	3 µg		12 ± 2	14 ± 4	41 ± 6	102 ± 7	51 ± 5
	TE	10 µg		14 ± 4	12 ± 3	38 ± 5	104 ± 4	51 ± 9
		33 µg		13 ± 1	15 ± 2	30 ± 4	99 ± 6	59 ± 13
		100 µg		13 ± 3	16 ± 1	33 ± 9	108 ± 9	53 ± 6
		333 µg		11 ± 3^P	20 ± 3^P	39 ± 4^P	101 ± 12^P	56 ± 5^P
		1000 µg		13 ± 5^P	11 ± 2^P	44 ± 11^P	103 ± 21^P	48 ± 6^P
		2500 µg		8 ± 2^P	13 ± 1^P	38 ± 8^P	76 ± 5^P	46 ± 7^P
		5000 µg		7 ± 2^P M	12 ± 2^P M	42 ± 2^P M	91 ± 5^P M	45 ± 6^P M
	2-AA	2.5 µg		577 ± 11	391 ± 15	3248 ± 386	3478 ± 18
	2-AA	10.0 µg						251 ± 18

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate	P M	Precipitate Manual count

 

 

Table2     Summary of Experiment II

Study Name: 1610101	Study Code: Harlan CCR 1610101
Experiment: 1610101 HV2 Pre	Date Plated: 20/03/2014
Assay Conditions:	Date Counted: 26/03/2014

 

Metabolic Activation	Test Group	Dose Level (per plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			12 ± 3	11 ± 2	22 ± 4	87 ± 11	45 ± 5
	Untreated			12 ± 4	10 ± 4	27 ± 6	108 ± 15	52 ± 6
	FAT 41044/A	10 µg		12 ± 2	9 ± 2	22 ± 7	82 ± 10	46 ± 7
	TE	33 µg		14 ± 4	7 ± 2	21 ± 2	85 ± 19	48 ± 8
		100 µg		12 ± 3	7 ± 2	25 ± 3	88 ± 12	51 ± 3
		333 µg		18 ± 7^P	11 ± 2^P	23 ± 4^P	88 ± 3^P	51 ± 4^P
		1000 µg		19 ± 3^P	7 ± 2^P	26 ± 4^P	76 ± 11^P	53 ± 4^P
		2500 µg		15 ± 6^P M	5 ± 1^P M	19 ± 1^P M	80 ± 3^P M	48 ± 2^P M
		5000 µg		7 ± 2^P M	4 ± 2^P M	18 ± 7^P M	81 ± 6^P M	43 ± 3^P M
	NaN3	10 µg		2892 ± 223			1995 ± 70
	4-NOPD	10 µg				286 ± 17
	4-NOPD	50 µg			60 ± 11
	MMS	2 µL						721 ± 22
With Activation	DMSO			25 ± 3	30 ± 0	44 ± 13	89 ± 11	61 ± 11
	Untreated			22 ± 7	25 ± 1	52 ± 7	86 ± 14	67 ± 6
	FAT 41044/A	10 µg		21 ± 4	35 ± 2	49 ± 5	86 ± 10	60 ± 10
	TE	33 µg		20 ± 5	36 ± 7	50 ± 1	82 ± 10	58 ± 7
		100 µg		23 ± 8	27 ± 3	46 ± 2	85 ± 20	53 ± 2
		333 µg		17 ± 3^P	33 ± 8^P	52 ± 3^P	76 ± 10^P	52 ± 4^P
		1000 µg		18 ± 5^P	30 ± 3^P	49 ± 6^P	71 ± 1^P	50 ± 2^P
		2500 µg		14 ± 8^P	27 ± 7^P	51 ± 3^P	55 ± 4^P	45 ± 7^P
		5000 µg		9 ± 2^P M	24 ± 6^P M	52 ± 10^P M	64 ± 14^P M	45 ± 9^P M
	2-AA	2.5 µg					1388 ± 33
	2-AA	2.5 µg		562 ± 16	201 ± 15
	2-AA	10 µg						792 ± 113
	Congo red	500 µg				262 ± 23

 

Key to Positive Controls		Key to Plate Postfix Codes
NaN3 2-AA 4-NOPD Congo red MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine Congo red methyl methane sulfonate	P M	Precipitate Manual count

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with and without rat- and hamster S9

In conclusion, it can be stated that during the described mutage¬nicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used with and without rat and hamster S9.

Executive summary:

This study was performed to investigate the potential of FAT 41044/A TE to induce gene muta­tions according to the plate incorporation assay with rat liver S9 (experiment I), and the pre-incubation test with hamster liver S9 (experiment II) using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

 

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate. The test item was tested at the following concentrations:

Pre-Experiment/Experiment I:   3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                            10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

 

The test item precipitated in the overlay agar in the test tubes from 100 to 5000 µg/plate in experiment I and from 1000 to 5000 µg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate in both experiments. The undissolved particles had no influence on the data recording.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred most of the strains. A minor reduction in the number of revertants (below the indication factor of 0.5) was observed in strains TA 1535 and TA 1537 in experiment I without S9 mix at 2500 µg/plate and in experiment II in strain TA 1537 in the absence of metabolic activation at 5000 µg/plate and in strain TA 1535 in the presence of metabolic activation at 5000 µg/plate.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with FAT 41044/A TE at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowled­ged border of biological relevance.

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease in induced revertant colonies.