2-[4,5-dihydro-4-methyl-4-(1-methylethyl)-5-oxo-1H-imidazol-2-yl]-3-pyridine carboxylate; imazapyr (ISO)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-03-05 to 2013-03-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimentelle Toxikologie und Ökologie, 67056 Ludwigshafen, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: COD-001701
- Expiration date of the lot/batch: 2010-02-01
- Purity test date: 2012-10-24

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (typically +25°C) or cooler
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed until 01 Nov 2014 as indicated by the sponsor.

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Cofactor-supplemented postmitochondrial fraction (S9) obtained from livers of rats treated with an enzyme-inducing agent (phenobarbital/beta-naphthoflavone)

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 2600, 5200 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: historical control data which demonstrated that DMSO is suitable

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

Remarks:

see details in results tables

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

see details in results tables

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

see details in results tables

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

see details in results tables

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 h to 72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Rationale for test conditions:

OECD 471 Guideline study test conditions

Evaluation criteria:

Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

Statistics:

Calculation of standard deviation, mean

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none occured
- Water solubility: none occured
- Precipitation: none occured
- Definition of acceptable cells for analysis: Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.
- Other confounding effects: none occured

RANGE-FINDING/SCREENING STUDIES: Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st Experiment.


HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above
- Negative (solvent/vehicle) historical control data: The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: detected by decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer

Any other information on results incl. tables

Table 1: Standard Plate test

Concentration µg/plate	TA 1535	TA 1535	TA 100	TA 100	TA 1537	TA 1537	TA 98	TA 98	E.coli WP2 uvrA	E.coli WP2 uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Solvent control	9	11	39	51	9	11	15	28	85	93
33 µg	8	10	37	60	9	9	16	27	73	83
100 µg	9	12	42	47	6	10	15	30	78	80
333 µg	8	9	47	47	10	13	12	21	78	90
1000 µg	11	13	47	51	4	9	15	35	83	88
2600 µg	10	15	26	35	5	7	16	23	68	79
5200 µg	7 (R*)	12 (R*)	20 (R*)	25 (R*)	4 (R*)	7 (R*)	12 (R*)	15 (R*)	63 (R*)	46 (R*)
MNNG	1071	-	923	-	-	-	-	-	-	-
2-AA	-	411	-	1464	-	224	-	1291	-	372
AAC	-	-	-	-	545	-	-	-	-	-
NOPD	-	-	-	-	-	-	837	-	-	-
4-NQO	-	-	-	-	-	-	-	-	740	-

R*: Reduced Background Growth

MNNG:N-methyl-N'-nitro-N-nitrosoguanidine

2-AA: 2-aminoanthracene

AAC: 9-aminoacridine

NOPD: 4-nitro-o-phenylendiamine

4-NQO: 4-Nitroquinoline-N-oxide

 

Table 2: Preincubation Test

Concentration µg/plate	TA 1535	TA 1535	TA 100	TA 100	TA 1537	TA 1537	TA 98	TA 98	E.coli WP2 uvrA	E.coli WP2 uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Solvent control	11	10	38	39	5	7	20	30	52	26
33 µg	10	12	39	40	5	7	19	27	55	28
100 µg	9	8	43	41	6	6	21	31	49	27
333 µg	9	10	37	42	7	4	21	32	54	21
1000 µg	14	8	40	45	7	5	15	23	58	25
2600 µg	6 (R*)	11 (R*)	20 (R*)	23 (R*)	3 (R*)	3 (R*)	8 (R*)	13 (R*)	18 (R*)	16 (R*)
5200 µg	R*	R*	16	R*	R*	R*	R*	R*	R*	8 (R*)
MNNG	1288	-	1278	-	-	-	-	-	-	-
2-AA	-	192	-	1092	-	201	-	1153	-	252
AAC	-	-	-	-	429	-	-	-	-	-
NOPD	-	-	-	-	-	-	893	-	-	-
4-NQO	-	-	-	-	-	-	-	-	516	-

R*: Reduced Background Growth

MNNG:N-methyl-N'-nitro-N-nitrosoguanidine

2-AA: 2-aminoanthracene

AAC: 9-aminoacridine

NOPD: 4-nitro-o-phenylendiamine

4-NQO: 4-Nitroquinoline-N-oxide