N-(4-chlorophenyl)-2-[(4-methyl-2-nitrophenyl)azo]-3-oxobutyramide

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:

2015

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:

2015

Report date:

2015

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

Reference substance name:

N-(4-chlorophenyl)-2-[(4-methyl-2-nitrophenyl)azo]-3-oxobutyramide

EC Number:

260-618-3

EC Name:

N-(4-chlorophenyl)-2-[(4-methyl-2-nitrophenyl)azo]-3-oxobutyramide

Cas Number:

57206-89-0

Molecular formula:

C17H15ClN4O4

IUPAC Name:

N-(4-chlorophenyl)-2-[(4-methyl-2-nitrophenyl)diazenyl]-3-oxobutanamide

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 in both experiments

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

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

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; 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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS Effects of pH:
- Water solubility: not soluble
- Precipitation: The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate in experiment I and from 1000 to 5000 µg/plate in experiment II. The undissolved particles had no influence on the data recording.
- Other confounding effects: none
COMPARISON WITH HISTORICAL CONTROL DATA: performed, no deviations
ADDITIONAL INFORMATION ON CYTOTOXICITY: No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabol¬ic activation.

Remarks on result:

other: other: reverse mutation assay

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Summary Tabellen

Table1     Summary of Experiment I

Study Name: 1722002	Study Code: Envigo 1722002
Experiment: 1722002 VV Plate	Date Plated: 27/10/2015
Assay Conditions:	Date Counted: 30/10/2015

 

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		9 ± 1		30 ± 12	183 ± 18	44 ± 5
	Untreated			8 ± 2		11 ± 6		32 ± 8	185 ± 24	37 ± 3
	N-(4-	3 µg		13 ± 4		7 ± 2		24 ± 7	185 ± 25	37 ± 6
	Chlorophenyl)-2-	10 µg		12 ± 4		7 ± 2		23 ± 1	162 ± 15	42 ± 7
	[(4-methyl-2-	33 µg		10 ± 5		11 ± 2		25 ± 3	144 ± 13	39 ± 9
	nitrophenyl)azo]-3-	100 µg		15 ± 2		12 ± 3		25 ± 8	170 ± 22	39 ± 10
	oxobutyramide	333 µg		13 ± 1^P		8 ± 2^P		33 ± 3^P	198 ± 21^P	40 ± 9^P
		1000 µg		15 ± 6^P		12 ± 2^P		29 ± 2^P	179 ± 9^P	38 ± 9^P
		2500 µg		12 ± 3^P M		8 ± 2^P		27 ± 6^P	164 ± 24^P M	32 ± 4^P M
		5000 µg		10 ± 2^P M		7 ± 1^P M		29 ± 6^P M	168 ± 11^P M	36 ± 3^P M
	NaN3	10 µg		1033 ± 89					1957 ± 71
	4-NOPD	10 µg						354 ± 52
	4-NOPD	50 µg				90 ± 10
	MMS	2.0 µL								898 ± 53
With Activation	DMSO			15 ± 3		10 ± 4		26 ± 8	179 ± 32	43 ± 13
	Untreated			13 ± 3		15 ± 6		33 ± 7	200 ± 10	52 ± 6
	N-(4-	3 µg		13 ± 1		11 ± 4		36 ± 5	179 ± 20	42 ± 10
	Chlorophenyl)-2-	10 µg		10 ± 3		10 ± 5		33 ± 13	165 ± 12	50 ± 7
	[(4-methyl-2-	33 µg		12 ± 2		11 ± 2		27 ± 6	164 ± 15	39 ± 8
	nitrophenyl)azo]-3-	100 µg		15 ± 3		14 ± 2		33 ± 3	190 ± 14	62 ± 6
	oxobutyramide	333 µg		15 ± 2^P		13 ± 3^P		33 ± 3^P	186 ± 25^P	57 ± 2^P
		1000 µg		11 ± 1^P M		10 ± 3^P		26 ± 6^P	167 ± 36^P	49 ± 14^P
		2500 µg		12 ± 2^P M		9 ± 3^P M		29 ± 6^P M	147 ± 24^P M	40 ± 4^P M
		5000 µg		10 ± 3^P M		6 ± 1^P M		27 ± 3^P M	114 ± 31^P M	34 ± 3^P M
	2-AA	2.5 µg		451 ± 28		178 ± 17		4275 ± 368	4600 ± 316
		10.0 µg								353 ± 67

 

 

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: 1722002	Study Code: Envigo 1722002
Experiment: 1722002 HV2 Pre	Date Plated: 04/11/2015
Assay Conditions:	Date Counted: 09/11/2015

 

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

10 ± 3

26 ± 5

131 ± 11

39 ± 3

Untreated

14 ± 5

9 ± 2

28 ± 2

156 ± 20

38 ± 6

N-(4-

3 µg

10 ± 5

10 ± 5

27 ± 1

129 ± 18

37 ± 4

Chlorophenyl)-2-

10 µg

13 ± 2

11 ± 6

32 ± 10

130 ± 13

33 ± 5

[(4-methyl-2-

33 µg

8 ± 2

11 ± 6

27 ± 5

159 ± 21

34 ± 4

nitrophenyl)azo]-3-

100 µg

10 ± 3

11 ± 4

28 ± 5

116 ± 3

35 ± 5

oxobutyramide

333 µg

15 ± 2

10 ± 6

28 ± 5

126 ± 13

35 ± 5

1000 µg

14 ± 6^P

10 ± 4^P

32 ± 6^P

149 ± 18^P

46 ± 5^P

2500 µg

16 ± 1^P M

11 ± 1^P M

34 ± 6^P M

133 ± 24^P M

37 ± 2^P M

5000 µg

18 ± 2^P M

10 ± 2^P M

26 ± 7^P M

127 ± 8^P M

32 ± 4^P M

NaN3

10 µg

1009 ± 58

2016 ± 136

4-NOPD

10 µg

412 ± 60

4-NOPD

50 µg

83 ± 6

MMS

2 µL

807 ± 31

With Activation

DMSO

21 ± 4

30 ± 3

40 ± 4

91 ± 7

59 ± 2

Untreated

26 ± 3

30 ± 3

44 ± 15

95 ± 16

69 ± 9

N-(4-

3 µg

23 ± 3

31 ± 4

43 ± 14

73 ± 25

61 ± 8

Chlorophenyl)-2-

10 µg

17 ± 6

30 ± 4

53 ± 3

85 ± 19

61 ± 3

[(4-methyl-2-

33 µg

20 ± 10

39 ± 7

56 ± 6

120 ± 30

59 ± 14

nitrophenyl)azo]-3-

100 µg

26 ± 11

45 ± 10

58 ± 4

91 ± 15

64 ± 1

oxobutyramide

333 µg

25 ± 3

40 ± 8

49 ± 7

65 ± 16

50 ± 13

1000 µg

17 ± 2^P

38 ± 7^P

41 ± 6^P

56 ± 6^P

45 ± 4^P

2500 µg

16 ± 2^P M

29 ± 10^P M

36 ± 7^P M

75 ± 5^P M

42 ± 3^P M

5000 µg

12 ± 2^P M

25 ± 3^P M

24 ± 2^P M

84 ± 6^P M

38 ± 3^P M

2-AA

2.5 µg

1217 ± 329

2-AA

2.5 µg

255 ± 49

189 ± 10

2-AA

10 µg

858 ± 41

Congo red

500 µg

426 ± 11

 

 

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

In conclusion, it can be stated that during the described mutagenicity 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.
Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of the test item to induce gene muta­tions according to the plate incorporation test (experiment I) with and without rat S9 mix and the pre-incubation test (experiment II) with and without hamster S9 mix using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, TA 100, and theEscherichia colistrain WP2 uvrA.

The assay was performed in two independent experiments. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations in both experiments:

3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

The test item precipitated in the overlay agar in the test tubes from 333 to 5000 µg/plate. Precipitation of the test item in the overlay agar on the incubated agar plates was observed from 333 to 5000 µg/plate in experiment I and from 1000 to 5000 µg/plate in experiment II. 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 in all strains with and without metabol­ic activation.

No substantial increase in revertant colony numbers of any of the fivetester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (rat and hamster 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 and showed a distinct in­crease of induced revertant colonies.