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Toxicological information

Genetic toxicity: in vitro

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD guideline 471 (adopted 1997)

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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(RCC-Cytotest Cell Research GmbH, Rossdorf, Germany)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Biofert Plusz
Details are presented in "Confidential details on test material"


Method

Target gene:
The Salmonella typhimurium histidine (his) and the E. coli tryptophan (trp) reversion system measures his- → his+ and trp- → trp+ reversions, respectively.
Species / strain
Species / strain:
other: S. typhimurium TA 1535, TA 100, TA 1537, TA 98; E. coli WP2uvrA
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix; male Wistar rat liver S9 prepared 24 h after termination of induction period (80 mg/kg b.w. Phenobarbital i.p. and 80 mg/kg bw ß-Naphthoflavone p.o. each on three consecutive days)
Test concentrations with justification for top dose:
Pre-experiment/experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate (related to dry mass)
Experiment II: 156, 312, 625, 1250, 2500, 5000 µg/plate (related to dry mass)
Vehicle:
- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: according to the solubility properties of the test item
Controls
Negative controls:
yes
Solvent controls:
yes
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: sodium azide (10 µg/plate in TA1535 & TA100), 4-nitro-o-phenylene-diamine (10 µg/plate in TA98 & 50 µg/plate in TA1537), methyl methane sulfonate (3 µL/plate in WP2uvrA);
Remarks:
with metabolic activation: 2-aminoanthracene (2.5 µg/plate in S. typhimurium and 10 µg/plate in E. coli)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 4 hours
- Exposure duration: 48 hours


SELECTION AGENT (mutation assays): minimal histidine agar

NUMBER OF REPLICATIONS: 3 plates per dose level in each experiment; 2 independent experiments


DETERMINATION OF CYTOTOXICITY
- Method: To evaluate the cytotoxicity of the test item, a pre-experiment was performed. Eight concentrations were tested for toxicity and mutation induction with three plates each.

Evaluation criteria:
The test item is considered as mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of 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.
Statistics:
According to OECD Guideline 471, a statistical analysis of data is not mandatory.

Results and discussion

Test results
Species / strain:
other: S. typhimurium TA 1535, TA 100, TA 1537, TA 98; E. coli WP2uvrA
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH value was specified as 4.4. No effects to this value were noted
- Water solubility: completely soluble in water
- Precipitation: none


RANGE-FINDING/SCREENING STUDIES: In a pre-experiment, strains TA 1535, TA 1037, TA 98, TA 100, and WP2 uvrA were tested at 8 concentartions with each 3 plates. Concentrations of the dry mass was between 3 and 5000 µg/plate. No toxic effects were observed and no mutagenic activity. Same results in experiment II.


COMPARISON WITH HISTORICAL CONTROL DATA: yes


ADDITIONAL INFORMATION ON CYTOTOXICITY: no cytotoxicity detected

Any other information on results incl. tables

The plates incubated with the test item showed normal background growth up to 5000 µg/plate of the dry mass with and without metabolic activation in both independent 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 af any of the five tester strains was observed following treatment at any concentration level, neither in the presenee nor absence of metabolic aetivation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally aeknowledged border of biological relevance.

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

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test item did not induce gene mutations in S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2uvrA with and without metabolic activation at concentrations up to 5 mg/plate (related to dry mass).