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Genetic toxicity: in vitro

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in vitro gene mutation study in bacteria
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov 2006 - June 2007
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
according to
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
Type of assay:
bacterial reverse mutation assay

Test material

Test material form:
solid: particulate/powder


Target gene:
The main genetic properties of these strains are:
Strain: his-Mutation: rfa: uvrB: pkM101:
TA97a D6610 yes yes yes
TA98 D3052 yes yes yes
TA100 G46 yes yes yes
TA102 G428 yes no yes
TA1535 G46 yes yes no
D6610 and D3052 are frameshift mutations, reversion can occur by addition or deletion of base-pairs. G46 is a base pair mutation, reversion occurs by the substitution of a single base. In G428 the codon CAA, coding for glutamine is mutated to the stop-codon TAA. The functionality of the wild type can be restored by base pair mutation as well as by deletion of 3 or 6 bases.
The rfa mutation leads to a reduced lipopolysaccharide barrier in the cell wall and allows larger molecules to pass the cell wall. Bacteria with this mutation are sensitive to crystal violet.
UvrB results in a loss of the DNA-excision repair system. This increases the sensitivity to mutagenic influences. Bacteria with this mutation are sensitive to UV light.
The plasmid pkM101 also disturbs the ability of the bacteria to repair genetic damage and
therefore increases their sensitivity to mutagens. Bacteria with this plasmid are resistant
against ampicillin. TA102 is also resistant against tetracycline.
Species / strain
Species / strain / cell type:
bacteria, other: Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535.
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from Aroclor 1254 induced microsomes of rat liver.

The microsomal fraction of homogenised livers of female Sprague Dawley rats treated once with 500 mg/kg of Aroclor 1254 was used. The preparation was done in December 2006.
Four days after treatment, the feed was withdrawn for one night. The livers of the animals were removed and homogenised in cold 0.15 mol/L KCl. Three mL of homogenate were obtained per gram of liver. Then the homogenate was centrifuged for 10 minutes at 9000 x g. The supernatant contained the microsomes. Small portions of the microsomes were stored in liquid nitrogen. Immediately before use they were thawed and mixed with the cofactor solution. The metabolic activity of the microsomes was verified by the positive control substances of each study.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 1667, 556, 185, 62 and 21 µg/plate
Concentration range in the main test (without metabolic activation): 1667, 556, 185, 62 and 21 µg/plate
Vehicle / solvent:
Solvent: Dimethylsulfoxide DMSO
Untreated negative controls:
Negative solvent / vehicle controls:
Positive controls:
Positive control substance:
sodium azide
other: 2-Aminoanthracene, 1,8-Dihydroxy-anthraquinone, 4-Nitro-o-phenylenediamine, t-Butyl-hydroperoxide
Details on test system and experimental conditions:
Concentration of the test substance resulting in precipitation: 556 µg/plate
Bacterial strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535, obtained from Prof. Bruce N. Ames, Berkeley, California, were used.
The bacteria were stored in small portions in a solution of 6 % DMSO in phosphate buffered saline in liquid nitrogen.
The actual batch of the strains was tested for ampicillin resistance (TA102: ampicillin/tetracycline resistance), UV-sensitivity and sensitivity against crystal violet, for spontaneous mutation frequencies and for sensitivities against the positive control substances in September 2000. The bacteria were stored frozen since that time.
Rationale for test conditions:
"SPP100 P7" was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECDguideline 471 and directive 2000/32/EC, part B.13/14.

The concentrations for the first experiment were set according to a preliminary toxicity test,
see #.
A precipitate was seen at 5000 μg/plate, which impeded the counting of the colonies. At 1667 μg/plate a slight precipitate was still observed in the plates, but the counting of the colonies was possible. It was therefore decided to use 1667 μg/plate as the highest concentration which would allow the evaluation of the plates. Each of the other 4 concentrations was 1/3 of the preceding one.

# Preliminary toxicity test
Different concentrations of test substance solutions were mixed with phosphate buffer or S9-mix, bacteria (TA100) and top-agar, as described in 3.5 and spread over a plate with minimal agar. The plates were incubated at 37 °C for 2 days and the growth of the bacterial background and the density of revertant colonies was determined.
Evaluation criteria:
Means and standard deviations were calculated for the number of mutants in every concentration group.
The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
• For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2½ fold of the amount of the spontaneous revertants.
• For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 12/3 fold of the amount of the spontaneous revertants.
These threshold values were derived from the variations in the control samples of our historic data of the Ames test.
Counting of colonies
The plates with less than about 50 revertant colonies, i.e. the plates of TA98 and TA1535
with the exception of the positive controls, were counted visually by marking the colonies with
a felt tipped pen. The other plates were photographed with a video camera and the picture
files were scanned for colonies by a computer program.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
> 1667 µg/plate
Cytotoxicity / choice of top concentrations:
> 1667 µg/plate
Vehicle controls validity:
Positive controls validity:
Additional information on results:
In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values was obtained. Metabolic activation did not change these results.
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)

Applicant's summary and conclusion

According to these results, "SPP100 P7" is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 1667 μg/plate.

negative with metabolic activation
negative without metabolic activation