Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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
Study period:
2008-11-07 to 2008-11-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well performed OECD and GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 - mix, rat
Test concentrations with justification for top dose:
33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Untreated control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO control
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium azide (NaN3), 4-nitro-o-phenylene-diamine (4-NOPD), Methyl methane sulfonate (MMS) ; each tested seperately
Remarks:
positive control without metabolic activation
Untreated negative controls:
yes
Remarks:
Untreated control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO control
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracen
Remarks:
positive control with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION:
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.
From the thawed ampoules of the strains 0.5 mL suspension was transferred into 250 mL Erlenmeyer flasks containing 20 mL nutrient medium. A solution of 20 pL ampicillin (25 pg/mL) was added to the strains TA 98 and TA 100.

This nutrient medium contains per litre:

8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt) 5 g NaCl (MERCK, D-64293 Darmstadt)

The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (108-109 cells/mL).

The plates with the selective agar were obtained from E. Merck, D-64293 Darmstadt.

The overlay agar contains per litre:

a) for Salmonella strains:
6.0 g MERCK Agar Agar, MERCK, D-64293 Darmstadt
6.0 g NaCl, MERCK, D-64293 Darmstadt
10.5mg L-Histidine×HCl×H2O, MERCK, D-64293 Darmstadt
12.2mg Biotin, MERCK, D-64293 Darmstadt

b) for Escherichia coli:
6.0 g MERCK Agar Agar, MERCK, D-64293 Darmstadt
6.0 g NaCl, MERCK, D-64293 Darmstadt
2.5 mg Tryptophan, MERCK, D-64293 Darmstadt

Sterilisations were performed at 121 °C in an autoclave.

Preparation of S9-Mix:
Phenobarbital/J3-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar HanIbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and J3-Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo(a)pyrene. The protein concentration in the S9 preparation was 36.2 mg/mL (lot no. R 010808). Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10% v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:

8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate 4 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al..

Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, if the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Dose Selection
In the pre-experiment the concentration range of the test item was 3 5000 pg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 pg/plate were chosen as maximal concentration.
The concentration range included two logarithmic decades. The following concentrations of the active ingredient were tested in experiment II:
33; 100; 333; 1000; 2500; and 5000 pg/plate

Experimental Performance
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100µL Test solution at each dose level (solvent or reference mutagen solution (positive control)
500µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
100µL Bacteria suspension
2000µL OVerlay agar


DURATION
In the pre-incubation assay 100 pL test solution (solvent or reference mutagen solution (positive control)), 500 pL S9 mix / S9 mix substitution buffer and 100 pL bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After pre- incubation 2.0 mL overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.



Evaluation criteria:
Evaluation of Results
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of
twice (strains TA 98, TA 100, and WP2 uvrA) or
thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed (3).

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration (2). An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

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
Remarks:
with and without S9
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occured in the test groups with and without metabolic activation
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
Remarks:
with and without S9
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occured in the test groups with and without metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the experimental conditions reported, the test item did not induce gene mutations.
Executive summary:

The test item 2-Benzimidazolol was assessed for its potential to induce gene mutations inthe plate incorporation test (experiment I) and the pre-incubation test (experiment II) usingSalmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escheri­chia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without livermicrosomal activation. Each concentration and the controls were tested in triplicate. Thetest item was tested at the following concentrations of the active ingredient:

Pre-Experiment/Experiment I:            3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:                                                       33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 pg/plate with and without S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains wasobserved following treatment with 2-Benzimidazolol at any dose level, neither in thepresence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generallyacknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­crease of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pairchanges or frameshifts in the genome of the strains used.