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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
- Name of test material (as cited in study report): Heliogen Blau K 6915
- Test substance No.: 00/0503-1
- Analytical purity: >= 98 g / 100 g
- Lot/batch No.: 00-0503-1
- Storage condition of test material: Room temperature

Method

Target gene:
Salmonella typhimurium: Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).
Escherichia coli: Determination of the rate of induced back mutations of bacterial mutant from trypthophan auxotrophy (trp-) to tryptophan prototrophy (trp+).
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from the liver of male Sprague-Dawley rats (treated i.p. with 500 mg/kg bw Aroclor 1254 5 days before sacrifice), mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer pH7.4).
Test concentrations with justification for top dose:
1st experiment:
Standard plate test with and without S9-mix (all strains): 0, 20, 100, 500, 2500 and 5000 µg per plate
2nd experiment:
Preincubation test with and without S9-mix (S. typhimurium T1535 and TA100): 0, 20, 100, 500, 2500 and 5000 µg per plate
3rd experiment:
Preincubation test with and without S9-mix (S. typhimurium TA1535, TA98 and E. coli WP2 uvrA): 0, 20, 100, 500, 2500 and 5000 µg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO had been demonstrated to be a suitable vehicle in bacterial reverse mutation tests; historical control data are available for DMSO.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: With S-9 mix: 2-aminoanthracene; without S-9 mix: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine
Details on test system and experimental conditions:
METHOD OF APPLICATION:
A standard plate test and a preincubation test were conducted, both with and without metabolic activation (S9 mix). Each test was conducted in triplicates.

STANDARD PLATE TEST:
The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et al. (Mut. Res. 31: 347-364, 1975) and Maron and Ames (Mut. Res. 113: 173-215, 1983)
Salmonella typhimurium:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42-45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation).
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar:
980 mL purified water
20 mL Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Escherichia coli:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation).
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.
The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M.H.L. and Muriel, W.J. (Mut. Res. 38: 3-32, 1976), with the exception of solution E (tryptophan solution), which has previously been added to the soft agar:
300 mL solution B (agar)
100 mL solution A (saline solution)
8 mL solution C (glucose solution)
10 mL solution D (casein solution)
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

PREINCUBATION TEST:
The experimental procedure is based on the method described by Yahagi et al. (Mut. Res. 48: 121-130, 1977) and Matsushima et al. (Factors modulating mutagenicity in microbial tests. In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York, 1980):
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.

CONTROLS:
Negative control:
Parallel with each experiment with and without S9-mix, negative controls (solvent control, sterility control) were carried out for each tester strain in order to determine the spontaneous mutation rate.
Positive controls:
The following positive control substances were used to check the mutability of the bacteria and the activity of the S9-mix. With S9 mix: 2-aminoanthracene, 2-AA (2.5 µg/plate for all 4 Salmonella strains, 60 µg/plate for E. coli strain); without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine, MNNG (5 µg/plate for TA 1535 and TA100); 4-nitro-o-phenylenediamine, NOPD (10 µg/plate for TA 98), 4-nitroquinoline-1-oxide, 9-Aminoacridine, AAC (100 µg/plate for TA 1537), 4-NQO (5 µg/plate for E. coli WP2 uvrA).

TITER DETERMINATION:
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.
In the standard plate test, 0.1 mL of the overnight cultures is diluted to 10E-6 in each case. Test tubes containing 2 mL portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL vehicle (without and with test substance)
0.1 mL fresh bacterial culture (dilution: 10E-6)
0.5 mL S9 mix
In the preincubation test, 0.1 mL of the overnight cultures is diluted to 10E-6 in each case. 0.1 mL vehicle (with and without test substance), 0.1 mL bacterial suspension and 0.5 mL S9 mix are incubated at 37°C for about 20 minutes using a shaker. Subsequently, 2 mL of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added.
After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.
Evaluation criteria:
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 are within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Acceptance criteria:
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls is within the range of the historical negative control data for each tester strain.
- The sterility control reveales no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induce 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 is >= 1x10exp+8/ml.

Results and discussion

Test resultsopen allclose all
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:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TOXICITY:
No bacteriotoxic effect was observed.

SOLUBILITY:
Test substance precipitation was found from about 100 µg/plate onward.

MUTAGENICITY:
An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system in any bacterial strain tested.

Any other information on results incl. tables

Table 1: Maximum revertants/plate and corresponding test concentrations in thestandard plate test:

    

Strain

Tested compound

Maximum revertants/plate [corresponding dose unit in µg/plate]

 

 

without S9-mix

with S9-mix

S. typhimurium TA1535

DMSO

21 ± 2

23 ± 2

Test substance

23 ± 4 [2500]

25 ± 2 [100]

Positive Control

639 ± 15 [5; MNNG]

136 ± 33 [2.5; 2-AA]

S. typhimurium TA100

DMSO

108 ± 6 

109 ± 3

Test substance

104 ± 4 [20]

112 ± 5 [500]

Positive Control

887 ± 73 [5; MNNG]

809 ± 59 [2.5; 2-AA]

S. typhimurium TA98

DMSO

33 ± 2 

44 ± 4

Test substance

34 ± 5 [100]

57 ± 8 [5000]

Positive Control

712 ± 33 [10; NOPD]

572 ± 46 [2.5; 2-AA]

S. typhimurium TA1537

DMSO

10 ± 2

10 ± 3

Test substance

9 ± 3 [20]

12 ± 4 [2500]

Positive Control

609 ± 100 [100; AAC]

95 ± 3 [2.5; 2-AA]

E. coli WP2 uvrA

DMSO

48 ± 7

45 ± 3

Test substance

45 ± 13 [20]

52 ± 11 [500]

Positive Control

734 ± 46 [5; 4-NQO]

228 ± 26 [60; 2-AA]

2-AA = 2-aminoanthracene  

MNNG = N-methyl-N'-nitro-N-nitrosoguanidine  

NOPD = 4-nitro-o-phenylenediamine      

AAC = 9-aminoacridine      

4-NQO = 4-nitroquinoline-N-oxide      

 

Table 2: Maximum revertants/plate and corresponding test concentrations in thepreincubation test:

    

Strain

Tested compound

Maximum revertants/plate [corresponding dose unit in µg/plate]

 

 

without S9-mix

with S9-mix

S. typhimurium TA1535

DMSO

17 ± 2

17 ± 1

Test substance

17 ± 2 [100]

16 ± 2 [20]

Positive Control

795 ± 40 [5; MNNG]

203 ± 6 [2.5; 2-AA]

S. typhimurium TA100

DMSO

106 ± 6

109 ± 7

Test substance

108 ± 6 [20]

107 ± 4 [20]

Positive Control

1094 ± 105 [5; MNNG]

889 ± 68 [2.5; 2-AA]

S. typhimurium TA98

DMSO

28 ± 5

31 ± 3

Test substance

30 ± 7 [20]

41 ± 4 [5000]

Positive Control

877 ± 68 [10; NOPD]

632 ± 63 [2.5; 2-AA]

S. typhimurium TA1537

DMSO

9 ± 2

9 ± 2

Test substance

9 ± 3 [100]

9 ± 1 [20]

Positive Control

436 ± 18 [100; AAC]

94 ± 5 [2.5; 2-AA]

E. coli WP2 uvrA

DMSO

38 ± 5

29 ± 5

Test substance

39 ± 4 [5000]

35 ± 6 [2500]

Positive Control

483 ± 102 [5; 4-NQO]

223 ± 7 [60; 2-AA]

2-AA = 2-aminoanthracene  

MNNG = N-methyl-N'-nitro-N-nitrosoguanidine  

NOPD = 4-nitro-o-phenylenediamine      

AAC = 9-aminoacridine      

4-NQO = 4-nitroquinoline-N-oxide      

Applicant's summary and conclusion

Conclusions:
According to the results of the present study, the test substance did not lead to an increase in the number of his revertant colonies either with or without S9-mix in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.