Registration Dossier

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:
April 16, 2007 - July 11, 2007
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2007-01-19
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Symrose
- Substance type: technical product
- Physical state: colourless, liquid
- Storage condition of test material: ambient temperature, dark and dry
No further details are given.

Method

Target gene:
the S. typhimurium histidine (his) system
Species / strainopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
The assay was performed in two independent experiments both with and without liver 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: 1, 3, 10, 33, 100, 333 and 1000 µg/plate
Vehicle:
Vehicle(s)/solvent(s) used: DMSO
The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methyl methane sulfonate (MMS)
Remarks:
3µL/plate
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Migrated to IUCLID6: 10µg/plate
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine (4-NOPD)
Remarks:
10µg/plate and 50 µg/plate
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA)
Remarks:
2.5 µg/plate and 10 µg/plate
Details on test system and conditions:
TESTSTRAINS:
Genotype:
TA98: his D 3052; rfa-; uvrB-; R-factor
TA100: his G 46; rfa-; uvrB-; R-factor
TA102: his G 428; rfa-; uvrB+; R-factor
TA1535: his G 46; rfa-; uvrB-: base-pair substitutions
TA1537: his C 3076; rfa-; uvrB-: frame shift mutations

METHOD OF APPLICATION: in agar (plate incorporation)
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 (negative control) 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 (cf. test system, pre-culture of the strains),
- 2000µL overlay agar

METABOLIC ACTIVATION:
S9-preparation
Phenobarbital/ß-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8-12 weeks old male Wistar Hanlbm rats, weight approx. 220-320 g induced by applications of 80mg/kg body weight Phenobarbital i.p. and ß-Naphthoflavone p.o. 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.

S9-mix
Before the experiment an appropiate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 was 10% v/v in the S9 mix. Cofactors were added to the S9 mix to reach the following concentrations in the S9 mix: 8mM MgCl2, 33mM KCl, 5mM Glucose-6-phosphate and 5mM NADP in 100mM sodium-ortho-phosphate-buffer, pH 7.4.

DURATION:
- Preincubation period: In the pre-incubation assay 100 µL test solution, 500 µL S9 mix / S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated 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 minimal agar plates.
- Exposure duration: After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark .
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

DATA RECORDING:
The colonies were counted using the Petri Viewer Mk2 (Perceptive Instruments Ltd, Suffolk CB 7BN, UK) with the software program Ames Study Manager. The counter was connected to an IBM AT compatible PC with printer which printed out both, the individual and mean values of the plates for each concentration together with standard deviations and enhancement factors as compared to the spontaneous reversion rates (see tables of results). Due to reduced background growth, the colonies were partly counted manually.
Evaluation criteria:
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

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 TA 102) 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.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test resultsopen allclose all
Species / strain:
other: see specification above
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used
Cytotoxicity:
yes
Remarks:
at concentrations >333 µg/plate in experiment I and >100 for TA1537 and TA98 in experiment II
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'.
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
at concentrations >333 µg/plate in experiment I and >100 in experiment II
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 substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Symrose at any concentration 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 acknowled¬ged border of biological relevance.
Appropriate reference mutagens were used as positive controls. They showed a distinct in-crease in induced revertant colonies.

Any other information on results incl. tables

Table 1: Reduced background growth was observed at the following concentrations (µg/plate)

Strain

Experiment I

Experiment II

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

333 - 5000

333 - 5000

333, 1000

333, 1000

TA 1537

333 - 5000

333 - 5000

100 - 1000

333, 1000

TA 98

333 - 5000

333 - 5000

100 - 1000

333, 1000

TA 100

333 - 5000

333 - 5000

333, 1000

333, 1000

TA 102

333 - 5000

333 - 5000

100 - 1000

333, 1000

Table 2: Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5) were observed at the following concentrations (µg/plate)

Strain

Experiment I

Experiment II

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

333 - 5000

333 - 5000

333, 1000

333, 1000

TA 1537

333 - 5000

333 - 5000

100 - 1000

333, 1000

TA 98

333 - 5000

333 - 5000

333, 1000

333, 1000

TA 100

333 - 5000

333 - 5000

333, 1000

333, 1000

TA 102

333 - 5000

333 - 5000

333, 1000

333, 1000

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

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 pair changes or frameshifts in the genome of the strains used. Therefore, Symrose is considered to be non-mutagenic in this reverse mutation assay.