Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 17 to August 21, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2000
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
312.5, 625, 1250, 2500 and 5000 µg/plate as active ingredient.
Freely soluble and non-toxic substance in preliminary test.
Vehicle / solvent:
Water
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthramine (2AM)
Remarks:
with S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
without S9 mix
Details on test system and experimental conditions:
Bacterial strains
The five strains of Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were supplied by B.N. Ames' Laboratory (University of Califomia, Berkeley, USA). The strain of Escherichia coli: WP2uvrA was supplied by S. Venitt's Laboratory (ICR, Sutton, England).
They are stored in a cryoprotective medium (1 ml nutrient broth and 0.09 ml dimethylsulfoxide) in liquid nitrogen.
The day before treatment, cultures were inoculated from frozen permanents: a scrape was taken under sterile conditions and put into approximately 6 ml of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37 °C for about 14 hours, to produce bacterial suspensions.
Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine. The strain of Escherichia coli contains one mutation in the tryptophan operon, resulting in a requirement for tryptophan.
In addition, to increase their sensitivity to mutagenic items, further mutations have been added:
- the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to large molecules that do not penetrate the normal bacteria cell wall,
- the uvr (uvrB for Salmonella typhimurium and uvrA for Escherichia coli) mutation is a deletion of a gene coding for the DNA excision repair system, which renders the bacteria unable to use this repair mechanism to remove the damaged DNA,
- the addition of the plasmid pKM 101 to strains TA 98, TA 100 and TA 102 enhances their sensitivity of detection to some mutagens,
- in the case of TA 102, the histidine mutation is located on the multicopy plasmid pAQ1.
The TA 1535, TA 100, TA 102 and WP2 uvrA strains are reverted by base-pair substitution mutagens and the TA 1537 and TA98 strains by frameshift mutagens. In addition, the TAl02 strain detects oxidative mutagens.

Metabolic activation system
The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function. S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route.
The S9 fraction was preserved in sterile tubes at -80 °C, until use.
The S9 mix was prepared at +4 °C immediately before use and maintained at this temperature until added to the overlay agar. The composition of S9 mix was as follows:
Ingredient Final concentration
Glucose-6-phosphate 5 mM
NADP 4 mM
KCl 33 mM
MgCl2 8 mM
Sodium phosphate buffer pH 7.4 100 mM
S9 fraction, batch No. 1565, protein concentration (38.8 mg/ml) 10 % v/v
water to volume

Experimental design
Treatment
The test item was tested in a preliminary test and two mutagenicity experiments.
The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the preincubation method.
The direct plate incorporation method was performed as follows: test item solution (0.1 ml), S9 mix when required or phosphate buffer pH 7.4 (0.5 ml) and bacterial suspension (0.1 ml) were mixed with 2 ml of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45 °C). After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.
The preincubation method was performed as follows: test item solution (0.1 ml), S9 mix (0.5 ml) and the bacterial suspension (0.1 ml) were incubated for 60 minutes at 37 °C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate.
After 48 to 72 hours of incubation at 37 °C, revertants were scored with an automatic counter (Cardinal counter. Perceptive Instmments, Suffolk CB9 7 BN, UK).

Preliminary toxicity test
To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100, TA 102 and WP2 uvrA strains, with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Mutagenicity experiments
In two independent experiments, using three plates/dose-level, each strain was tested, with and without S9 mix, with:
- at least five dose-levels of the test item,
- the vehicle control,
- the appropriate positive control.
The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100, TA 102 and WP2 uvrA strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Results and discussion

Test results
Species / strain:
other: all strains
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary toxicity test
The test item was freely soluble in the vehicle (water for injections) at 50 mg/ml. Consequently, with a treatment volume of 100 µl/plate, the dose-levels were 10, 100, 500, 1000,2500 and 5000 µg/plate.
No precipitate was observed in Petri plates when scoring the revertants at all dose-levels.
A red coloration of agar was observed when scoring the revertants at all dose-levels.
No noteworthy toxicity was noted towards the four strains used, with and without S9 mix.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.
Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level selected for the main test was 5000 µg/plate, according to the criteria specified in the guidelines.
No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels.
Except for some sporadic decreases in the number of revertants noted in the second experiment mainly in the TA 98 strain, no toxicity was noted towards all the strains used, both with and without S9 mix.
In the TA 1537 strain in the first experiment with S9 mix, a 3.1-fold increase in the number of revertants was noted at 1250 µg/plate. This very slight increase was not considered as relevant since it was neither dose-related, nor reproducible and was attributed to the very low vehicle control value (mean value of 4 which is in the low side of the historical range).
The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the six strains.

Applicant's summary and conclusion

Conclusions:
No mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium and Escherichia coli.
Executive summary:

Methods

A preliminary toxicity test was performed to define the dose-levels of test substance to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, i.e. S9 mix prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37 °C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA98, TA100 and TA 102 and one strain of Escherichia coli: WP2 uvrA were used. Each strain was exposed to five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37 °C, the revertant colonies were scored.

Evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Test item was dissolved in water for injections. All concentrations and dose-levels were expressed as active item, taking into account a purity of 87.5 %.

The dose-levels of the positive controls were as follows:

without S9 mix:

- 1 µg/plate of sodium azide (NaNs): TA 1535 and TA 100 strains,

- 50 µg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

- 0.5 µg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

- 0.5 µg/plate of Mitomycin C (MMC): TA 102 strain,

- 2 µg/plate of 4-nitroquinoline 1-oxide (4-NQO): WP2 uvrA strain.

with S9 mix:

- 2 µg/plate of 2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains,

- 10 µg/plate of 2-Anthramine (2AM): TA 102 and WP2 uvrA strains.

Results

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level selected for the main test was 5000 µg/plate, according guidelines.

The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate, for both mutagenicity experiments with and without S9 mix.

No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. No significant toxicity nor increase in the number of revertants was noted in all the strains used, both with and without S9 mix.