[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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

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 test material (as cited in study report): Gelb LD 6259 (No 08/0646-1)
- Physical state: solid/ sun yellow
- Analytical purity: Main component: 87.6 area% or 89.1 area% (depending on the method; see analytical report, study code 08L00346)
- batch No.: 0817 VP 01
- Stability under test conditions: the stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility
- Storage condition of test material: room temperature; avoid temperatures > 60°C
- Other: the test substance was homogeneous by visual inspection

Method

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction is prepared according to Ames et al. from male Wistar rats [Crl:WI(Han)] (200 - 300 g) treated with 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally

Test concentrations with justification for top dose:

1st Experiment (standard plate test): 0, 20, 100, 500, 2500 and 5000 μg/plate;
2nd experiment (preincubation test): 0, 312.5, 625, 1250, 2500 and 5 000 μg/plate;
3rd experiment (standard plate test):0, 23, 115, 575, 2875 and 5750 μg/plate;
4th experiment (preincubation test): 0, 23, 115, 575, 2875 and 5750 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: to achieve a solution of the test substance in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly.
All test substance formulations were prepared immediately before administration.

Controls

Untreated negative controls:

yes

Remarks:

sterile controls were also done

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: All dissolved in DMSO; with S9 mix: 2-aminoanthracene (2-AA; 2.5 μg/plate, for TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate for E. coli WP2 uvrA). Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; 5 μg/plate for TA 1535, TA 100), see below

Remarks:

positive control without S-9 mix, continue: 4-nitro-o-phenylenediamine (NOPD; 10 μg/plate for TA 98), 9-aminoacridine (AAC; 100 μg/plate for TA 1537 and 4-nitroquinoline-N-oxide (4-NQO; 5 μg/plate for E. coli WP2 uvrA).

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Expression duration: 48-72 hours in the dark at 37°C

NUMBER OF REPLICATIONS: 4 experiments in triplicates

DETERMINATION OF CYTOTOXICITY
- Method: colonie count. The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: after incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Evaluation criteria:

Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, five doses of the test substance are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the 1st Experiment. 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.
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.
- The titer of viable bacteria was > 108/mL.
Assessment criteria
The test chemical 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.
A test substance is generally considered non-mutagenic in this test if:
- The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Statistics:

not applicable

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

No increase in the number of his+ or trp+ revertants neither in standard plate test nor in preincubation test (see Table 1&2)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

A weak bacteriotoxic effect (slight decrease in the number of his+ revertants) was occasionally observed in the standard plate test and the preincubation assay depending on the strain and test conditions from about 2 875 μg/plate onward.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No test substance precipitation was found with and without S9 mix

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in four experiments carried out independently of each other (standard plate test and preincubation assay). Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Table 1: Standard Plate Test; Maximum revertants per plate and corresponding concentration

 

Strain	Experiment	Dose level (µg/plate)	Maximum revertants per plate (mean of triplicate) at the corresponding dose
			Without S-9 mix			With S-9 mix
			Mean	SD	FAC	Mean	SD	FAC
TA 1535	First	Negative control	16	3	1.0	17	1	1.0
		20, 500	17	2	1.1
		100				16	2	0.9
		Positive Control	586	105	35.9	119	2	6.9
	Second	Negative control	14	3	1.0	16	2	1.0
		115				20	1	1.2
		575	20	7	1.4
		Positive Control	532	23	37.1	149	40	9.1
TA 100	First	Negative control	95	9	1.0	100	16	1.0
		20	107	17	1.1
		100				99	11	1.0
		Positive Control	586	32	6.2	662	95	6.6
	Second	Negative control	14	6	1.0	16	2	1.0
		115				20	1	1.2
		575	20	7	1.4
		Positive Control	532	23	37.1	149	40	9.1
TA 98	First	Negative control	28	2	1.0	34	5	1.0
		2500				32	10	1.0
		5000	26	3	0.9
		Positive Control	462	41	16.3	534	21	15.9
	Second	Negative control	32	7	1.0	38	4	1.0
		23	28	5	1.0	34	6	1.0
		Positive Control	437	30	13.8	787	25	20.5
TA 1537	First	Negative control	7	1	1.0	8	2	1.0
		100				13	2	1.5
		5000	10	2	1.3
		Positive Control	350	20	47.7	86	12	10.3
	Second	Negative control	8	2	1.0	8	1	1.0
		23				10	1	1.0
		5750	10	4	1.3
		Positive Control	344	45	43.0	152	37	18.2
E. coli WP2 uvrA	First	Negative control	35	4	1.0	55	2	1.0
		2500	39	3	1.1
		5000				53	5	1.0
		Positive Control	872	18	24.7	252	10	4.6
	Second	Negative control	34	5	1.0	38	7	1.0
		23	39	5	1.1
		44				44	4	1.2
		Positive Control	649	124	20.3	223	5	5.9
FAC: factor

 

Table 2: Preincubation Test; Maximum revertants per plate and corresponding concentration

 

Strain	Experiment	Dose level (µg/plate)	Maximum revertants per plate (mean of triplicate) at the corresponding dose
			Without S-9 mix			With S-9 mix
			Mean	SD	FAC	Mean	SD	FAC
TA 1535	First	Negative control	16	5	1.0	17	2	1.0
		312.5	19	3	1.1
		5000				16	2	0.9
		Positive Control	572	15	35.0	148	16	8.9
	Second	Negative control	15	2	1.0	16	3	1.0
		23				15	3	0.9
		5750	16	4	1.1
		Positive Control	1076	23	73.4	127	15	7.8
TA 100	First	Negative control	98	4	1.0	98	3	1.0
		625	103	6	1.0	104	5	1.1
		Positive Control	864	25	8.8	817	60	8.3
	Second	Negative control	128	9	1.0	112	19	1.0
		115				148	4	1.3
		575	135	17	1.1
		Positive Control	1199	8	1.1	809	53	7.2
TA 98	First	Negative control	30	3	1.0	34	5	1.0
		312.5				39	2	1.2
		2500	31	3	1.0
		Positive Control	553	20	18.6	502	164	14.9
	Second	Negative control	30	4	1.0	29	5	1.0
		23	32	6	1.1	34	2	1.2
		Positive Control	477	67	15.9	621	36	21.4
TA 1537	First	Negative control	8	1	1.0	8	3	1.0
		312.5				9	3	1.2
		5000	10	5	1.3
		Positive Control	393	32	51.2	140	16	18.2
	Second	Negative control	7	3	1.0	9	2	1.0
		115				39	2	1.2
		2500	31	3	1.0
		Positive Control	553	20	18.6	502	164	14.9
E. coli WP2 uvrA	First	Negative control	30	2	1.0	45	5	1.0
		312.5	38	8	1.3	48	9	1.1
		Positive Control	576	23	19.0	226	21	5.0
	Second	Negative control	41	3	1.0	53	8	1.0
		2875	52	5	1.3	50	8	0.9
		Positive Control	535	45	13.1	242	22	4.6
FAC: factor