[No public or meaningful name is available]

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:

2008-06-19 till 2008-09-02

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:

other: Commission Directive 2000/32/EC, L1362000, Annex 4DU, dated May 19, 2000.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines: “Kanpoan No. 287 -- Environment Protection Agency“ “Eisei No. 127 -- Ministry of Health & Welfare“ “Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry“

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Details on test material:

- public name of test material:
Reaction mass of 6,13-dichloro-3,10-bis{[2-({[(2-chloroethyl)sulfonyl]alkanoyl}amino)ethyl]-amino}- polycarboheterocyclo 4,11-disulfonic acid, mono and/or disodium salt and 6,13-dichloro-3-{[2-({[(2-chloroethyl)sulfonyl]alkanoyl}amino) ethyl]amino}-10-[(2-{[4-(ethenylsulfonyl)alkanoyl]amino}ethyl)amino] polycarboheterocyclo -4,11-disulfonic acid, mono and/or di sodium salt
- Physical state: solid, dark blue powder
- Analytical purity: approx. 86.6%
- Lot/batch No.: VER 2108 BOP 02/07
- Expiration date of the lot/batch: November 30, 2012

Method

Target gene:

see Table 1 (any other information on material and methods)

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

Metabolic activation system:

Phenobarbital/-Naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate and 5000 mg/plate of the active ingredient
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate and 5000 µg/plate of the active ingredient

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:
Deionised water. The solvent was chosen because of its solubility properties.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

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

Details on test system and experimental conditions:

METHOD OF APPLICATION:
plate incorporation and preincubation

DURATION
Preincubation period: 30 min at 30ºC

Exposure duration: at least 48h at 37 ºC in the dark

SELECTION AGENT (mutation assays): Histidine (Salmonella) resp. Tryptophane (E.coli)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

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),
2000 μL Overlay agar
According to the pre-incubation method (second experiment) 100 μL test solution, solvent or positive control, 500 μL S9 mix / S9 mix substitution buffer, and 100 μL bacteria suspension were mixed in a test tube and incubated at 30°C for 30 minutes . After preincubation 2.0 ml overlay agar (45°C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.

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:

not applicable (no toxicity)

Results and discussion

Test results

Test results 1

Species / strain:

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

Positive controls validity:

valid

Test results 2

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none recorded
- Evaporation from medium: not expected, very low vapor pressure
- Water solubility: 60.4 g/L
- Precipitation:none recorded


RANGE-FINDING/SCREENING STUDIES: no

COMPARISON WITH HISTORICAL CONTROL DATA: yes, data in the range of the historical control


ADDITIONAL INFORMATION ON CYTOTOXICITY: none

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Pre-Experiment/Experiment I (plate incorporation)

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	Deionised water		17 ± 3	11 ± 2	28 ± 6	144 ± 2	55 ± 13
	Untreated		10 ± 4	11 ± 2	32 ± 4	146 ± 8	48 ± 7
	FAT 40841/A TE	3 μg	14 ± 6	17 ± 3	32 ± 4	139 ± 19	48 ± 7
		10 μg	13 ± 4	12 ± 3	31 ± 9	147 ± 6	58 ± 8
		33 μg	14 ± 1	16 ± 1	34 ± 6	134 ± 5	59 ± 3
		100 μg	15 ± 1	14 ± 2	33 ± 9	142 ± 11	56 ± 6
		333 μg	12 ± 2	12 ± 3	30 ± 9	136 ± 16	41 ± 9
		1000 μg	11 ± 4 D M	11 ± 3 D M	28 ± 3 D M	129 ± 6 D M	40 ± 7 D M
		2500 μg	13 ± 1 D M	10 ± 2 D M	30 ± 3 D M	139 ± 7 D M	41 ± 4 D M
		5000 μg	10 ± 2 D M	10 ± 4 D M	28 ± 6 D M	134 ± 7 D M	39 ± 4 D M
	NaN3	10 μg	1713 ± 106		489 ± 63	1735 ± 101
	4-NOPD	10 μg		115 ± 13
	4-NOPD	50 μg
	MMS	3.0 μl					1141 ± 56
With Activation	Deionised water		18 ± 5	22 ± 5	45 ± 6	149 ± 8	71 ± 15
	Untreated		23 ± 4	25 ± 2	49 ± 9	141 ± 3	69 ± 2
	FAT 40841/A TE	3 μg	18 ± 1	23 ± 5	45 ± 8	138 ± 8	63 ± 2
		10 μg	19 ± 2	21 ± 2	35 ± 4	152 ± 8	67 ± 4
		33 μg	19 ± 2	18 ± 3	39 ± 1	148 ± 13	62 ± 16
		100 μg	19 ± 6	18 ± 5	39 ± 10	132 ± 16	62 ± 10
		333 μg	14 ± 1	15 ± 1	46 ± 4	127 ± 14	60 ± 7
		1000 μg	12 ± 2 D M	15 ± 3 D M	35 ± 3 D M	128 ± 7 D M	52 ± 2 D M
		2500 μg	13 ± 2 D M	14 ± 3 D M	31 ± 5 D M	124 ± 6 D M	49 ± 5 D M
		5000 μg	11 ± 1 D M	15 ± 3 D M	31 ± 4 D M	124 ± 5 D M	47 ± 3 D M
	2-AA	2.5 μg	268 ± 10	292 ± 25	1647 ± 50	2365 ± 67
	2-AA	10.0 μg					306 ± 30

D Densely coloured plate

M Manual count

Pre-Experiment/Experiment Ia (Pre-incubation) 

( 5000 μg calculated on the active ingredient)

Metabolic Activation	Test Group	Dose Level (Mg/plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA1537	TA 98 TA	TA 100	100 WP2 uvrA
Without Activation	Deionised water		16 ± 7	9 ± 3	24 ± 7	128 ± 7	46 ± 13
	Untreated		16 ± 5	13 ± 4	32 ± 5	139 ± 3	51 ± 6
	FAT 40841/A TE	5000 μg	15 ± 4 D M	10 ± 4 D M	27 ± 7 D M	128 ± 18 D M	47 ± 4 D M
	NaN3	10 μg	1846 ± 27			1791 ± 64
	4-NOPD	10 μg			418 ± 18
	4-NOPD	50 μg		97 ± 9
	MMS	3 μL					1228 ± 21
With Activation	Deionised water		22 ± 4	15 ± 2	42 ± 6	147 ± 11	59 ± 4
	Untreated		20 ± 1	16 ± 4	42 ± 2	148 ± 9	67 ± 4
	FAT 40841/A TE	5000 μg	17 ± 4 D M	13 ± 3 D M	35 ± 10 DM	145 ± 14 D M	61 ± 5 D M
	2-AA	2.5 μg				1867 ± 92
			255 ± 18	227 ± 5	1421 ± 246
	2-AA	10 μg					275 ± 20

Key to Positive Controls Key to Plate Postfix Codes

NaN3 sodium azide D Densely coloured plate

2-AA 2-aminoanthracene M Manual count

4-NOPD 4-nitro-o-phenylene-diamine

MMS methyl methane sulfonate

* repeated experiment

Experiment II (Pre-Incubation)

Test Group	Dose Level (μg/plate)	Revertant Colony Counts (Mean ±SD)
		TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Deionised water		18 ± 3	16 ± 3	27 ± 4	146 ± 4	49 ± 7
Untreated		16 ± 5	13 ± 4	30 ± 3	167 ± 35	50 ± 1
FAT 40841/A TE	33 μg	17 ± 5	17 ± 2	33 ± 7	146 ± 12	52 ± 4
	100 μg	18 ± 4	13 ± 6	28 ± 5	150 ±   5	54 ± 6
	333 μg	15 ± 2	16 ± 3	26 ± 3	130 ± 10	56 ± 1
	1000 μg	20 ± 6 D M	13 ± 2 D M	22 ± 3 D M	137 ± 5 D M	46 ± 2 D M
	2500 μg	17 ± 2 D M	11 ± 2 D M	26 ± 3 D M	130 ± 4 D M	48 ± 6 D M
	5000 μg	19 ± 4 D M	9 ± 3 D M	26 ± 1 D M	127 ± 10 D M	37 ± 4 D M
NaN3	10 μg	1929 ± 64			1887 ± 107
4-NOPD	10 μg			399 ± 44
4-NOPD	50 μg		109 ± 11
MMS	3.0 μl					339 ± 41
Deionised water		17 ± 5	30 ± 8	46 ± 11	128 ± 23	45 ± 3
Untreated		19 ± 5	27 ± 4	42 ± 4	119 ± 16	50 ± 2
FAT 40841/A TE	33 μg	18 ± 5	32 ± 2	47 ± 4	135 ± 21	54 ± 11
	100 μg	17 ± 2	24 ± 4	39 ± 5	121 ± 15	48 ± 9
	333 μg	17 ± 4	31 ± 3	45 ± 2	120 ± 18	46 ± 4
	1000 μg	17 ± 4 D M	25 ± 2 D M	43 ± 2 D M	116 ± 8 D M	41 ± 4 D M
	2500 μg	19 ± 3 D M	26 ± 2 D M	47 ± 7 D M	125 ± 8 D M	47 ± 4 D M
	5000 μg	15 ± 2 D M	24 ± 2 D M	31 ± 2 D M	115 ± 6 D M	47 ± 3 D M
2-AA	2.5 μg				624 ± 82
	2.5 μg	756 ± 90	230 ± 46
2-AA	10.0 μg					233 ± 57
Congo Red				911 ± 183

Experiment IIa (Pre-Incubation) 

( 5000 μg FAT 40841/A TE calculated on the active ingredient)

Metabolic Activation	Test Group	Dose Level (Mg/plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA1537	TA 98 TA	TA 100	100 WP2 uvrA
Without Activation	Deionised water		22 ± 6	9 ± 5	25 ± 7	152 ± 11	46 ± 12
	Untreated		15 ± 6	16 ± 4	35 ± 3	140 ± 3	50 ± 7
	FAT 40841/A TE	5000 μg	22 ± 2 D M	8 ± 2 D M	27 ± 8 D M	143 ± 8 D M	47 ± 6 D M
	NaN3	10 μg	1873 ± 53			1805 ± 35
	4-NOPD	10 μg			420 ± 11
	4-NOPD	50 μg		104 ± 8
	MMS	3 μL					1219 ± 20
With Activation	Deionised water		24 ± 6	21 ± 4	43 ± 7	137 ± 11	58 ± 2
	Untreated		31 ± 6	20 ± 5	42 ± 6	148 ± 8	67 ± 5
	FAT 40841/A TE	5000 μg	24 ± 6 D M	20 ± 5 D M	44 ± 2 DM	140 ± 18 D M	57 ± 3 D M
	2-AA	2.5 μg				870 ± 15
			472 ± 15	93 ± 12
	2-AA	10 μg					290 ± 31
	Congo Red	500 μg			1401 ± 246

Key to Positive Controls Key to Plate Postfix Codes

NaN3 sodium azide D Densely coloured plate

2-AA 2-aminoanthracene M Manual count

4-NOPD 4-nitro-o-phenylene-diamine

MMS methyl methane sulfonate

* repeated experiment

Applicant's summary and conclusion

Conclusions:

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

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, test material TE is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

Test material was tested according to OECD Guideline 471.

It is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

This study was performed to investigate the potential of test material 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 the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments with and without liver microsomal activation. Due to irregular bacteria growth, no data were evaluated in experiment I from strains TA 1537 and TA 98. This part was repeated under identical conditions (reported as part of experiment I). Additional experiments were performed with the highest concentration (5000 Mg/plate) calculated on the active ingredient. The plate incorporation assay with and without rat S9 mix is reported as experiment Ia and the preincubation assay with and without hamster S9 mix is reported as experiment IIa. Each concentration, including the controls, was 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 and 5000 µg/plate of the active ingredient Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate and 5000 µg/plate of the active ingredient

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both experiments.

No toxic effects, evident as a reduction in the number of revertants, were observed with and without metabolic activation in both experiments.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test material at any dose 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 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 not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, test material is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.