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Toxicological information

Genetic toxicity: in vitro

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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:
From April 12 to 25 August, 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to internationally accepted testing guidelines and performed according to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell lines (if applicable):
TA 1537, genotype his C 3076; rfa-; uvrB; frame shift mutations.
TA 98, genotype his D 3052; rfa-; uvrB; R-factor; frame shift mutations.
TA 1535, genotype his G 46; rfa-; uvrB; base-pair mutations.
TA 100, genotype his G 46; rfa-; uvrB; R-factor; base-pair mutations.
WP2, genotype trp; trp; uvrA; base-pair substitutions and others
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
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
Vehicle:
On the day of the experiment, the test item was dissolved in deionised water. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
No precipitation of the test item occurred up to the highest investigated dose.
Controls
Negative controls:
yes
Remarks:
Concurrent untreated.
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine and 2-aminoanthracene
Remarks:
Without met. act: sodium azide for TA 1535, TA 100; 4-NOPD for TA 1537, TA 98; MMS for WP2 uvrA. With met. act: 2-M for TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA.
Details on test system and conditions:
PRECULTURE
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 µl ampicillin (25 µg/ml) was added to the strains TA 98 and TA 100.
This nutrient medium contains per litre:
8 9 Merck Nutrient Broth (MERCK, 0-64293 Darmstadt)
5 9 NaCI (MERCK, 0-64293 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 (10^8-10^9 cells/ml).

Selective Agar: the plates with the selective agar were obtained from E. Merck, 0-64293 Darmstadt.
Overlay Agar: the overlay agar contains per litre:
for Salmonella strains:
6.0 g MERCK Agar Agar*
6.0 g NaCI*
10.5 mg L-Histidine x HCl x H2O*
12.2 mg Biotin*
For Escherichia coli:
6.0 g MERCK Agar Agar*
6.0 g NaCI*
2.5 mg Tryptophan*
* (MERCK, 0-64293 Darmstadt)
Sterilisations were performed at 121 °C in an autoclave.

S9 PREPARATION
Phenobarbital/β-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old maleWistar Hanlbm rats, weight approx. 220-320 g induced by applications of 80 mg/kg b.w. Phenobarbital Lp. (Oesitin; 0-22335 Hamburg) and β-Naphthoflavone p.o. (Aldrich, 0-89555 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 KCI 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 31.4 mg/ml.

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 KCI
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath.

PRE-EXPERIMENT 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 µg/plate. The pre-experiment is reported as experiment I. Since-no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration.

TEST 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:
100 µl test solution at each dose level (solvent or reference mutagen solution (positive control))
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
100 µl Bacteria suspension (cf. test system, pre-culture of the strains)
2000 µl Overlay agar
In the pre-incubation assay 100 µl test solution (solvent or reference mutagen solution (positive control)), 500 µl S9 mix / S9 mix substitution buffer and 100 µl bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After preincubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates.
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.
Evaluation criteria:
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.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
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 results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
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 was observed following treatment with test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

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

Any other information on results incl. tables

Summary of Results Pre-Experiment and Experiment I

Metabolic Activation Test-Group Dose level (µg/plate) Revertant Colony Counts (Mean ± SD)
TA1535 TA1537 TA98 TA100 WP2 uvr/A

Without activation

Deionised water 27 ± 7 9 ± 0 32 ± 3 132 ± 5 46 ± 5
Untreated 36 ± 9 9 ± 1 29 ± 6 124 ± 7 43 ± 2
Test item 3 µg 38 ± 3 8 ± 1 25 ± 5 125 ± 2 55 ± 6
10 µg 31 ± 6 12 ± 4 34 ± 8 129 ± 13 55 ± 11
      33 µg 39 ± 10 12 ± 2 32 ± 13 135 ± 10 48 ± 4
100 µg 40 ± 0 9 ± 1 33 ± 6 149 ± 11 51 ± 12
333 µg 32 ± 7 14 ± 2 28 ± 6 140 ± 10 53 ± 7
1000 µg 25 ± 1 9 ± 1 31 ± 4 136 ± 10 49 ± 2
2500 µg 32 ± 7 9 ± 2 32 ± 4 124 ± 16 49 ± 7
5000 µg 32 ± 3 12 ± 2 29 ± 8 132 ± 16 55 ± 3
NaN3 10 µg 3129 ± 34 3103 ± 85
4-NOPD 10 µg 367 ± 15
4-NOPD 50 µg 68 ± 16
MMS 3.0 µl 1217 ± 46

With activation

Deionised water 32 ± 7 14 ± 2 37 ± 18 144 ± 19 85 ± 1
Untreated 29 ± 2 14 ± 2 40 ± 3 138 ± 10 72 ± 11
Test item 3 µg 26 ± 4 14 ± 2 34 ± 2 144 ± 16 67 ± 4
10 µg 35 ± 13 13 ± 3 32 ± 7 150 ± 5 68 ± 4
33 µg 36 ± 8 11 ± 3 40 ± 5 139 ± 13 58 ± 12
100 µg 34 ± 8 13 ± 3 36 ± 6 147 ± 11 57 ± 6
333 µg 33 ± 12 13 ± 7 29 ± 2 141 ± 13 62 ± 13
1000 µg 27 ± 3 20 ± 10 29 ± 5 138 ± 8 60 ± 2
2500 µg 27 ± 5 14 ± 7 33 ± 5 142 ± 8 65 ± 8
5000 µg 21 ± 1 10 ± 2 32 ± 1 148 ± 4 66 ± 20
2-AA 2.5 µg 298 ± 29 203 ± 13 1522 ± 58 1968 ± 159
2-AA 10.0 µg 203 ± 27

NaN3: sodium azide

2 -AA: 2-aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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 pair changes or frameshifts in the genome of the strains used.
Executive summary:

Method

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Eschericchia coli strain WP2 uvrA, according to the OECD guideline 471.

The assay was performed in two independent experiments both with and without microsomal activation. Each concentration and the controls were tested in triplicate. The test item was tested at the following concentrations:

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

Results

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

No toxic effects, evident as a reduction in the number of revertants occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

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 pair changes or frameshifts in the genome of the strains used.