1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-(C16-18(even numbered) and C18 unsaturated acyl) derivs., hydroxides, inner salts

Genetic toxicity: in vitro

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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:

from 15 August 2002 to 14 October 2002

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:

2003

Report date:

2003

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:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries. Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production bureau, November 24, 2000

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:

- Name of test material (as cited in study report): DV6850
- Substance type: powder
- Physical state: solid
- Analytical purity: 83.7%
- Lot/batch No.: R0332-52C
- Expiration date of the lot/batch: 2003/09/30
- Storage condition of test material: ca 4°C in the dark in dry conditions

Method

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 pKM 101

Metabolic activation:

with and without

Metabolic activation system:

supplemented liver homogenate fraction (S9 mix)

Test concentrations with justification for top dose:

test 1 (range-finding): 5, 15, 50, 150, 500, 1500, 5000 µg/plate
test 2: 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The solubility of DV6850 was assessed at 50 mg/mL in water, in which it dissolved and formed a clear gel after sonication for 30 minutes. Water (purified in-house by reverse osmosis) was, therefore, used as the solvent for this study.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Used in the absence of S9 mix

Controls 2

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Used in the absence of S9 mix

Controls 3

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Used in the absence of S9 mix

Controls 4

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2)

Remarks:

Used in the absence of S9 mix

Controls 5

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

Used in the presence of S9 mix

Controls 6

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Used in the presence of S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, test 1) and preincubation (30 min pre-incubation, test 2)

DURATION
- Preincubation period: 30 minutes in second test, no preincubation in first test
- Exposure duration: ca. 72 hours

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no

Evaluation criteria:

If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice the concurrent solvent/vehicle controls with some evidence of a positive dose-relationship (increased revertant colony counts at concentrations below that at which the maximal increase is obtained), it will be considered to show evidence of mutagenic activity in this test system. No statistical analysis was performed.
If exposure to a test substance does not produce an increase in revertant colony numbers in two separate experiments, with any bacterial strain either in the presence or absence of S9 mix, it will be considered to show no evidence of mutagenic activity in this test system. No statistical analysis was performed.

Statistics:

If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, even after the additional testing outlined in the mutation test procedure, the test data may have been subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are ususally analysis of variance followed by Dunnett's test. Biological significance should always be considered along with statistical significance.

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxic effects were observed at 5000 µg/mL without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

MAIN ASSAYS:
No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to DV6850 at any concentration in either the presence or absence of S9 mix.
No visible thinning of the background lawn of non-revertant cells was obtained following exposure to DV6850.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mean revertant colony counts for the solvent controls were within the 99% confidence limits of the current historical control range of the laboratory.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 - Results for test 1 (range-finding)

Metabolic activation	Test group	Dose level [µg/plate, for water: mL/plate]	Revertant colony counts (mean±SD)
			TA98	TA100	TA1535	TA1537	WP2uvrA
yes	DV6850	5000	57±5	161±13	17±3	26±2	214±54
yes	DV6850	1500	66±5	169±5	18±7	27±5	180±28
yes	DV6850	500	55±15	182±16	14±4	26±5	181±17
yes	DV6850	150	57±2	172±21	14±5	21±4	186±4
yes	DV6850	50	62±13	165±13	16±3	24±6	199±16
yes	DV6850	15	61±16	158±10	17±3	27±9	169±22
yes	DV6850	5	59±8	174±14	20±2	23±7	166±15
yes	water	0.1	60±10	192±27	24±3	19±8	152±8
no	DV6850	5000	38±12	150±11	26±3	13±5	127±43
no	DV6850	1500	41±4	156±14	22±4	18±3	203±14
no	DV6850	500	44±12	149±4	19±2	24±2	184±40
no	DV6850	150	52±4	155±16	10±4	10±3	194±26
no	DV6850	50	56±7	145±6	14±10	10±4	159±11
no	DV6850	15	54±11	148±6	22±9	7±2	169±7
no	DV6850	5	50±14	119±18	22±6	13±5	135±56
no	water	0.1	46±8	157±12	19±4	22±6	199±20
yes	Benzo(a)pyrene	5	666±87	743±8		350±8
yes	2-Aminoanthracene	2			65±5
yes	2-Aminoanthracene	10					601±563
no	2- Nitrofluorene	1	208±14
no	Sodium azide	0.5		588±32	405±15
no	9-Aminoacridine	30				273±88
no	AF-2	0.05					841±80

Table 2 - Results for test 2, with pre-incubation

Metabolic activation	Test group	Dose level [µg/plate, for water: mL/plate]	Revertant colony counts (mean±SD)
			TA98	TA100	TA1535	TA1537	WP2uvrA
yes	DV6850	5000	42±7	201±18	12±4	42±10	206±14
yes	DV6850	1500	39±11	187±31	23±1	42±12	140±56
yes	DV6850	500	49±13	161±11	19±3	31±3	215±9
yes	DV6850	150	47±5	173±16	21±2	23±12	211±36
yes	DV6850	50	51±12	191±21	17±2	49±10	232±21
yes	water	0.1	49±12	180±2	20±13	30±3	206±11
no	DV6850	5000	38±5	187±14	23±6	30±6	118±10
no	DV6850	1500	36±7	199±15	15±6	31±3	166±16
no	DV6850	500	33±4	182±21	20±5	27±3	136±3
no	DV6850	150	35±5	193±23	14±5	29±6	157±7
no	DV6850	50	39±4	172±12	14±4	24±5	162±27
no	water	0.1	34±10	173±18	12±3	31±7	135±23
yes	Benzo(a)pyrene	5	616±32	583±57		136±82
yes	2-Aminoanthracene	2			354±219
yes	2-Aminoanthracene	10					1149±119
no	2- Nitrofluorene	1	204±59
no	Sodium azide	0.5		369±49	111±79
no	9-Aminoacridine	30				690±175
no	AF-2	0.05					1566±121

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

It is concluded that, under the test conditions employed, DV6850 showed no evidence of mutagenic activity in this bacterial system.

Executive summary:

The mutagenic potential of DV6850 in a bacterial system was assessed in a valid GLP study. The study was conducted in compliance with the OECD Guideline 471: Bacterial Reverse Mutation Test.

In this in vitro assessment of the mutagenic potential of DV6850, histidine dependent auxotrophic mutants of Salmonella typhimurium, strains TA 1535, TA 1537, TA98 and TA100, and a tryptophan dependent mutant of Escherichia coli, strain WP2uvrA/pKM101 (CM891), were exposed to DV6850 diluted in water. Water was also used as a negative control.

Two independent mutation tests were performed in the presence and absence of liver preparations from Aroclor 1254—treated rats (S9 mix). The first (range-finding) test was a standard plate incorporation assay, the second involved a pre-incubation stage.

Concentrations of DV6850 up to 5000 µg/plate were tested. This is the standard limit concentration recommended in the regulatory guidelines that this assay follows. Other concentrations used were a series of ca half-log10 dilutions of the highest concentration. Slight cytotoxic effects were observed for E. coli treated at 5000 µg/mL without metabolic activation. No other signs of toxicity were observed towards the tester strains in either mutation test.

No evidence of mutagenic activity was seen at any concentration of DV6850 in either mutation test. The concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

It is concluded that, under the test conditions employed, DV6850 showed no evidence of mutagenic activity in this bacterial system.