Black HM 2482

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:

From March 5th to May 6th, 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 84/449

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

400-920-6

EC Name:

-

Cas Number:

89857-06-7

Molecular formula:

C50 H53 N11 O14 S

IUPAC Name:

5'-[2-(7-{2-[4-(2-{1'-[3-(dimethylazaniumyl)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-8-hydroxy-6-sulfonatonaphthalen-2-yl)diazen-1-yl]-6'-hydroxy-3,4'-dimethyl-2'-oxo-1',2'-dihydro-1λ⁵-[1,3'-bipyridin]-1-ylium bis(2-hydroxypropanoate)

Method

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and TA 1538

Details on mammalian cell type (if applicable):

obtained from B.N. Ames, University of California, Berkeley CA, 94720, USA, May 1981.

Metabolic activation:

with and without

Metabolic activation system:

LIVER MICROSOMAL FRACTION S-9 MIX
The S-9 was prepared on the day of the experiment.
Specific pathogen-free male Wistar rats (180 g - 300 g, KFM-Ha, outbred), received a single i.p. injection of Aroclor 1254 dissolved in Oleum Arachidis (200 mg/ml) at a dosage of 500 mg/kg of body weight to induce liver microsomal enzyme activity five days before the experiment.
The rats were killed by cervical dislocation and exsanguinated on the fifth day post application following a 14-16 hours starvation period.
Livers were removed under aseptic conditions and homogenized with ice-cold 0.15 molar potassium chloride (5 g of liver to 15 g of potassium chloride). The homogenates were centrifuged at 900 x g for 10 minutes at 0 to 2 °C. The supernatant fraction (S-9 fraction) was collected for the preparation of S-9 mix.

Composition of 1 ml S-9 mix:
Na2HPO4 100 µM
MgCl2 8 µM
KCl 33 µM
NADP 4 µM
G-6-P 5 µM
S-9 Fraction 0.3 ml

Test concentrations with justification for top dose:

The test article was investigated at the following concentrations: 1.58, 5, 15.8, 50, 158, 500, 1580, 5000 micrograms per plate (mcg/pl)

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

methylmethanesulfonate

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other:

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Characterization of strains:
The strains used are mutants derived from SaImonelIa typhimurium LT2 and have following genotype:
S.typhimurium TA 1535 his G46 rfa- uvrB-
S.typhimurium TA 1537 his C3076 rfa- uvrB-
S.typhimurium TA 1538 his D3052 rfa- uvrB-
S.typhimurium TA 98 his D3052 rfa- uvrB- R+
S.typhimurlum TA 100 his G46 rfa- uvrB- R+
AII the strains carry a mutation known as deep rough (rfa) which affects the cell membrane so that the normaI lipopolysaccharide cell coat is defective. These strains aIIow a greater permeability of the test agents across this membrane and into the cell.
In bacteria, almost aIl the primary damage caused to DNA by a mutagen is repaired by excision and recombination repair systems.Therefore, only a small percentage of the potential mutagenic damage is expresed. The tester strains used lack this excision repair system (uvrB), thus making them more sensitive to mutagens.
The strains TA 98 and TA 100 also carry an ampicillin resistance factor (R) which makes them particularly sensitive to rnutagens. The strains TA 1535 and TA 100 are sensitive to base-pair
substitution mutagens; the strains TA 1537, TA 1538 and TA 98eare sensitive to frameshift- mutagens. The tester strains are checked at regular intervals for their genetic markers.

Storage:
The strain cultures are stored in sterile 0 5 ml ampouIes (0 . 45 ml bacteriaI culture and 0 . 05 ml Dimethylsulfoxide) at -7O degrees centrigrade.

Pre-culture of strains:
The bacteria were grown in a shaking water bath for 16 hours overnight at 37 degrees centigrade in 2.5% Nutrient Broth No. 2. After centrifugation, the bacteria were resuspended to a concentration of approximately 1 x 10 exp. 8 to 2 x l0 exp. 9 ceIls per milliliter in 0.16% Nutrient Broth and 0.5% sodium chloride. The concentration of germs was controlled photometrically and
determined in an experimental test with Histidine-rich potassium chloride solution on selective agar plates.

Study procedure
For each strain and dose IeveI, including the controls, a minimum of three plates where used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl Test- solution at each dose IeveI, solvent {negative control) or reference mutagen
solution (positive control),
500 µl S-9 mix (for test with metabolic activation) or 0.15 M NaHPO4, (for test without
metabolic activation),
100 µl Bacteria suspension (cf. test system, pre-culture of the strains),
2 ml Overlay agar consisting of 0.6% Bacto agar and 0.6% NaCl supplemented with 10% 0.5
mM L-Histidine and 0.5 mM Biotine solution.
The plates where incubated at 37 °C in the dark for 3 days. The his+ revertant colonies where counted with an Artek B80 counter or counted by hand if precipitation of test article precluded automatic counting. For the aseptic control experiment, 100 µl of S-9 mix where added to 2 mm overlay agar and treated as mentioned above.
For each test, the germ concentration of the bacterial strains was determined by pouring a mixture of 100 µl of a 2 x 10 exp.5 diluted bacteria suspension (overnight culture) with a Histidine-rich potassium chloride solution onto a selective agar plate. After three days of incubation at 37 °C the colonies where counted and the counts multiplied with the dilution factor.

TOXICITY TEST
To estimate the cytotoxicity of the test article, prototrophic bacteria (spontaneous revertants of TA 1537 = RTA) where added as an internal standard to the seIective agar pIates, together with
TA 1537 (cf . study procedure). The diference in the number of colonies on plates with and without added prototrophic bacteria at each dose level of the test article was compared to the
number of colonies obtained with the negative control. The ratio of the two values was expressed as relative survival rate with the negative control at a 100% survival rate.
AdditionaIIy, the toxicity of the test article can be shown in the mutagenicity assay, by a reduction in the number of revertants or by a reduction or a clearing of the background lawn.

Evaluation criteria:

The number of revertant colonies was recorded manually for each plate from the counter.

Statistics:

A material is identified as a mutagen in this test system if there is a reproducible demonstration of a dose effect relation with a 2-fold increase in the number of revertants over the controls in a minimum of one strain. With the strain TA 100, a 1.5-fold increase is the criterion for a positive result.
These criteria are generally in accordance with the international used standards for the evaluation of results obtained from the Ames test.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium 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

True negative controls validity:

not valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not valid

Positive controls validity:

valid

Any other information on results incl. tables

TOXICTY OF THE TEST CONTROL
In the toxicity experiment with test item, neither quantitative or qualitative evidence of a cytotoxic effect was observed. Precipitates in the agar occurred starting at 1580 mcg/pl. In the second experiment at the highest concentration of 5000 mcg/pl. the number of revertants was counted manually, and in TA 98 also at a concentration of 1580 mcg/pl.

CONTROL EXPERIMENTS
Control plates with the solvent (negative control) showed numbers of spontaneous revertant colonies per plate which were within the normal range of those cited in Ames et al (*).
Control plates with reference mutagens (positive controls) showed a distinct increase of the revertant colonies with the tester strains. This confirms the reversion properties of each strain. The positive results of the mutagens 2-Amino-anthracene and Benzo(a)pyrene indicate that the metabolizing system was functioning .
The aseptic control showed no contamination of the S-9 mix.

TEST WITH TEST ITEM
In the descRibed bacterial mutagenicity tests, no relevant increase of the revertant colony numbers was obtained in the Salmonella typhimurium strains used at all dose levels tested when compared with the corresponding controls. The presence of liver microsomal activation did not influence these findings. These results were confirmed in a second, independent experiment.
Slight, non-significant increases of revertants as detected in TA 100 without (1. experiment) or with (2. experiment) liver microsomal activation, and in TA 1537 with activating (2. experiment) are sporadic and considered to occur randomly.

 

* Ames, B.N., Mc Cann, J. and Yamasaki, E., Methods for detecting carcinogens with the Salmonella/mammalian-microsome mutagenicity test: Mutation Res. 31, 347 -364 .

Applicant's summary and conclusion

Conclusions:

In the present mutagenicity test and under the experimental conditions reported, the test article induced no point mutations by base-pair changes of frameshifts in the genome of the strains used.

Executive summary:

The test item was evaluated for mutagenicity in Bacterial Reverse Mutation Test, according to OECD guideline 471 (1983). Experiments were carried out using S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. The test was performed with and without liver microsomal activation. The test article was tested at the following concentrations: 1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 micrograms per plate. Each concentration, including the controls, was tested in triplicate. No toxic effect of the test article was observed. Up to the highest investigated dose, no relevant incerase of the revertant colony numbers was obtained in any Salmonella typhimurium strain used when compared with the corresponding controls. The presence of liver microsomal activation did not influence these findings.
These results were confirmed in a second, indipendent experiment.