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:
19 March, 2015 - 16 April, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study is conducted according to OECD TG 471, in compliance with GLP, without deviations that influence the quality of the results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

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:
(2008)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material in report: Floral Super
- Description: Clear liquid
- Storage condition of test material: At room temperature flushed with nitrogen

Method

Target gene:
- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene
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:
Rat liver S9-mix induced by Aroclor 1254.
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by Aroclor 1254.
Test concentrations with justification for top dose:
Direct plate:
- Dose range finding test:
TA 100 and WP2uvrA (without and with S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
- Experiment 1:
Due to cytotoxicity (in TA 100), the following dose levels were used:
TA 1535, TA 1537 and TA 98 (without S9): 0.54, 1.7, 5.4, 17, 52 and 164 μg/plate
TA 1535, TA 1537 and Ta 98 (with S9): 1.7, 5.4, 17, 52, 164 and 512 μg/plate

Preincubation:
- Dose range finding test:
TA 100 (without S9): 0.17, 0.54, 1.7, 5.4, 17, 52 and 164 µg/plate
TA 100 (with S9): 0.54, 1.7, 5.4, 17, 52, 164 and 512 µg/plate
WP2uvrA (without and with S9): 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
- Experiment 1:
Due to cytotoxicity (in TA 100), the following dose levels were used:
TA 1535, TA 1537, TA 98 and TA 100 (without S9): 0.056, 0.18, 0.55, 1.7, 5.4, 17 and 52 μg/plate
TA 1535, TA 1537 and TA 98 (with S9): 0.55, 1.7, 5.4, 17, 52 and 164 μg/plate
Vehicle:
- Solvent used: DMSO
- Justification for choice of solvent: the test substance was found to be soluble in DMSO up to 5000 µg/plate
Controls
Negative controls:
no
Solvent controls:
yes
Remarks:
(100 µL/plate)
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and conditions:
METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: (independent repeat): preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation
Evaluation criteria:
A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

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
Cytotoxicity:
yes
Remarks:
Toxicity was observed in all tester strains.
Vehicle controls valid:
yes
Positive controls valid:
yes
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Only in the preincubation assay without S9-mix at the concentration of 164 µg/plate and upwards.
Vehicle controls valid:
yes
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the direct plate assay precipitation (oily droplets) was observed at the concentration of 1600 and 5000 µg/plate. In the preincubation assay, precipitation was observed at concentrations of 512 μg/plate in tester strain TA100 and at 5000 μg/plate in tester strain WP2uvrA.

RANGE-FINDING/SCREENING STUDIES:
- Direct plate assay: In strain TA 100 toxicity (microcolonies) was observed at the concentration of 164 µg/plate and above, both in the absence and presence of S9. In tester strain WP2uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.
- Preincubation assay: In strain TA 100 toxicity was observed at the concentration of 164 µg/plate and above, in the presence of S9. In the absence of S9, toxicity was observed at concentration of 17 and 52 µg/plate. In tester strain WP2uvrA, toxicity was observed at the concentration of 164 µg/plate and above, in the absence of S9. In the presence of S9, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Direct plate assay: In all strains toxicity (microcolonies) was observed at the higher doses (at >= 164 µg/plate), both in the absence and/or presence of S9.
- Preincubation assay: In all strains toxicity (microcolonies) was observed at the concentrations of 17 and 52 µg/plate, in the absence of S9. In the presence of S9, in all strains toxicity was observed at the concentration of 164 µg/plate.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Floral Super is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay performed according to OECD 471 (1997) and GLP principles.
Executive summary:

The mutagenic activity of Floral Super was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiments: at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix. The dose levels were selected based on observed cytotoxicity in dose range finding tests in strain TA 100 (direct plate -/+ S9 and preincubation +S9 >= 164 µg/plate, preincubation -S9 17 and 52 µg/plate). Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and E. coli tester strain (WP2uvrA), both in the absence and presence of S9 -metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that Floral Super is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay.