2-((E)-(4-((4-(1,3-dioxo-1,3-dihydro-2H-hetereocarbapolycyclo-2-yl)alkyl)(ethyl)amino)-2-methyl)diazenyl)-5-nitro-1,3-dicarbonitrile, polyphenyl

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

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: "Commission Directive 2000/32/EC, L1362000, Annex 4D", dated May 19, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Name of the test material (as cited in the study report: FAT 40842/A TE
- Substance type: coloring dye
- Physical state: solid, dark bluish green powder
- Analytical purity: 96%
- Lot/batch No.: Blau DRI 2098 Op 1/07
- Expiration date of the lot/batch: June 30, 2014
- Storage condition of test material: at room temperature at about 20 ºC

Method

Target gene:

see Table 1 "any other information on materials and methods incl. tables"

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Test concentrations with justification for top dose:

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

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:The solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: NaN3 = sodium azide 4-NOPD = 4-Nitro-o-phenylene-diamine MMS = methyl methane sulfonate 2-AA = 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;
For each strain and dose level, including the controls three plates were used. The following materials were mixed in a test tube and poured onto the selective agar plates (first experiment):
- 100 μl Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500 μl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 μl Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000μl Overlay agar
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.


Rat S9 Mix
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.(2).
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.(2) and Prival and Mitchell (1).
1. Prival. M.J. and V.D. Mitchell (1982)
Analysis of a method for testing azo dyes for mutagenic activity in Salmonella
typhimurium in the presence of flavin mononucleotide and hamster liver S9
Mutation Res. 97, 103-116
2. Ames, B.N., W.E. Durston, E. Yamasaki, and F.D. Lee (1973)
Carcinogens are mutagens: a simple test system combining liver homogenates for
activation and bacteria for detection
Proc. Natl. Acad. Sci. (USA) 70, 2281-2285

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 (3).
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (2).
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:

No statistical evaluation of the data is required

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no significant effects by test item
- Effects of osmolality: no significant effects by test item
- Evaporation from medium: no
- Water solubility: low, suspended homogeniously in DMSO
- Precipitation: Precipitation of the test item was observed either in the overlay agar in the test tubes at 5000 Lg/plate and on the incubated agar plates from 333 Lg/plate up to 5000 Lg/plates.
- Other confounding effects: no


COMPARISON WITH HISTORICAL CONTROL DATA:
attached to the report


ADDITIONAL INFORMATION ON CYTOTOXICITY: no

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: reverse mutation assay

Any other information on results incl. tables

- Pre-Experiment/Experiment I

Metabolic Activation	Test Group	Dose Level (µg/plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO			23 ± 10	16 ± 5	33 ± 10	137 ± 17	49 ± 4
	Untreated			25 ± 5	13 ± 4	35 ± 8	148 ± 10	57 ± 15
	FAT 40842/ATE	3 µg		18 ± 5	14 ± 3	35 ± 13	127 ± 7	47 ± 6
		10 µg		16 ± 4	16 ± 1	35 ± 11	127 ± 2	46 ± 3
		33 µg		21 ± 7	18 ± 10	37 ± 8	152 ± 19	47 ± 3
		100 µg		23 ± 8	17 ± 2	45 ± 3	143 ± 12	52 ± 10
		333 µg		23 ± 3^P	16 ± 2^P	39 ± 7^P	146 ± 6^P	51 ± 17^P
		1000 µg		20 ± 7^P	19 ± 4^P	48 ± 2^P	154 ± 10^P	44 ± 4^P
		2500 µg		16 ± 1^P	20 ± 8^P	61 ± 9^P	146 ± 24^P	51 ± 14^P
		5000 µg		18 ± 3^P	17 ± 2^P	52 ± 13^P	158 ± 4^P	45 ± 4^P
	NaN3	10 µg		2313 ± 42			2080 ± 71
	4-NOPD	10 µg				405 ± 17
	4-NOPD	50 µg			110 ± 10
	MMS	3.0 µL						1226 ± 92
With Activation	DMSO			23 ± 4	23 ± 10	48 ± 13	142 ± 13	67 ± 3
	Untreated			22 ± 5	15 ± 2	40 ± 2	149 ± 19	71 ± 10
	FAT 40842/ATE	3 µg		33 ± 10	31 ± 6	166 ± 21	178 ± 5	70 ± 6
		10 µg		25 ± 8	37 ± 3	173 ± 22	164 ± 25	55 ± 3
		33 µg		22 ± 5	29 ± 6	170 ± 22	163 ± 15	69 ± 9
		100 µg		19 ± 8	24 ± 4	160 ± 26	165 ± 4	47 ± 6
		333 µg		22 ± 2^P	22 ± 5^P	161 ± 10^P	166 ± 15^P	48 ± 2^P
		1000 µg		22 ± 4^P	19 ± 2^P	132 ± 10^P	164 ± 8^P	58 ± 5^P
		2500 µg		13 ± 2^P	21 ± 1^P	155 ± 9^P	170 ± 11^P	65 ± 2^P
		5000 µg		17 ± 4^P	25 ± 7^P	156 ± 11^P	180 ± 16^P	65 ± 6^P
	2-AA	2.5 µg		235 ± 15	226 ± 12	1558 ± 66	1628 ± 515
	2-AA	10.0 µg						379 ± 133

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	Precipitate

- Experiment I a

Metabolic Activation	Test Group	Dose Level (µg/plate)		Revertant Colony Counts (Mean ±SD)
				TA 98
Without Activation	DMSO			27 ± 7
	Untreated			40 ± 4
	FAT 40842/ATE	3 µg		35 ± 2
		10 µg		36 ± 5
		33 µg		39 ± 2
		100 µg		46 ± 6
		333 µg		59 ± 7^P
		1000 µg		61 ± 6^P
		2500 µg		63 ± 2^P
		5000 µg		66 ± 5^P M
	4-NOPD	10 µg		482 ± 38
With Activation	DMSO			38 ± 3
	Untreated			38 ± 4
	FAT 40842/ATE	3 µg		131 ± 10
		10 µg		133 ± 11
		33 µg		143 ± 7
		100 µg		157 ± 19
		333 µg		165 ± 14^P
		1000 µg		172 ± 23^P
		2500 µg		161 ± 5^P
		5000 µg		176 ± 7^P M
	2-AA	2.5 µg		1683 ± 52

Key to Positive Controls		Key to Plate Postfix Codes
4-NOPD 2-AA	4-nitro-o-phenylene-diamine 2-aminoanthracene	P M	Precipitate Manual count

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation
positive without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strain TA 98 with and without of metabolic activation.
Therefore, FAT 40842/A TE is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The present study (Sokolowski 2009) was performed to investigate the potential of FAT 40842/A TE to induce gene mutations according to the plate incorporation assay with rat liver S9 (experiment I) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed with and without liver microsomal activation. A confirmatory experiment was performed with and without rat S9 mix with strain TA 98 (reported as experiment Ia). Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations:

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

The plates incubated with the test item showed normal background growth up to 5000 Lg/plate with and without metabolic activation. No toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation.

A moderate but dose dependent increase in revertant colony numbers was observed following treatment with FAT 40842/A TE in strain TA 98 with and without rat S9 mix.

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

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did induce gene mutations by frameshifts in the genome of strain TA 98 with and without of metabolic activation.

Therefore, FAT 40842/A TE is considered to be mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.