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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 July 2018 - 30 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sebacohydrazide
EC Number:
213-126-8
EC Name:
Sebacohydrazide
Cas Number:
925-83-7
Molecular formula:
C10H22N4O2
IUPAC Name:
sebacohydrazide
Test material form:
solid: particulate/powder
Details on test material:
Test item name (as stated in report): SDH
Test item storage: at room temperature protected from light

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate) induced by Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate, tested in strains TA100 and WP2uvrA, with and without S9-mix
First experiment: 52, 164, 512, 1600 and 5000 μg/plate, tested in strains TA1535, TA1537 and TA98, with and without S9-mix
Second experiment: 52, 164, 512, 1600 and 5000 μg/plate, tested in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA, with and without S9-mix
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Controls
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2-aminoanthracene (2AA)
Remarks:
Details on positive control substances in tables 1 and 2
Details on test system and experimental conditions:
TEST SYSTEM
- Salmonella typhimurium bacteria and Escherichia coli bacteria.
- Source: Trinova Biochem GmbH, Germany [Master culture from Dr. Bruce N. Ames (TA1535, TA1537, TA98, TA100; and Master culture from The National Collections of Industrial and Marine Bacteria, Aberdeen, UK (WP2uvrA)]

DOSE FINDING TEST
- Strains TA100 and WP2uvrA, with and without S9-mix. Eight concentrations were tested: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate, in triplicate.

EXPERIMENTAL DESIGN
Application in agar plates, in triplicate (3 plates per dose)
- Agar plates: 25 uL glucose agar medium
- Top agar: Bacteriological agar (Oxoid LTD) 0.6% w/v supplemented with sodium chloride (Merck) 0.5% w/v

METHODS
Experiment 1: Direct plate assay
- The dose finding test is part of this experiment. In the second part of this experiment the test item was tested with and without S9 in the strains TA1535, TA1537 and TA98
- 0.1 mL of bacterial culture (10^9 cells/mL), 0.1 mL of test item diluted in DMSO and either 0.5 ml S9-mix or 0.5 mL 0.1 M phosphate buffer to 3 mL of melted top agar and poured in the agar plates
- Agar plates were incubated at 37.0 ± 1.0°C for 48 ± 4 h

Experiment 2: Pre-incubation assay
- 0.1 mL of bacterial culture (10^9 cells/mL), 0.1 mL of test item diluted in DMSO and either 0.5 ml S9-mix or 0.5 mL 0.1 M phosphate buffer were pre-incubated for 30 ± 2 minutes by 70 rpm at 37 ± 1°C
- After pre-incubation, the solutons were added to 3 mL of melted top agar, mixed and poured in the agar plates
- Agar plates were incubated at 37.0 ± 1.0°C for 48 ± 4 h

INCUBATION
- Exposure duration: 48 ± 4 h (in the dark at 37.0 ± 1.0 °C)

NUMBER OF REPLICATIONS
- Each dose was tested in triplicate

DETERMINATION OF TOTOXICITY
- Observation of reduction of the bacterial background lawn, increase in the size of the microcolonies and the reduction of the revertant colonies

COLONY COUNTING
- Automatic counting: Sorcerer colony counting
- When precipitation of the test substance is observed, colonies are counted manually

ACCEPTABILITY CRITERIA
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated
Rationale for test conditions:
Recommended test system in international guidelines (e.g. OECD, EC)
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent control
b) The negative response should be reproducible in at least one follow up experiment

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three 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
Statistics:
No formal hypothesis testing was done

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
observed in direct plate assay, with and without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DIRECT PLATE ASSAY
- Precipitation: present at the start of the incubation period at the concentrations of 512 μg/plate and upwards in TA1535, TA1537 and TA98
- No precipitate was observed at the end of the incubation period
- Mutagenicity: no increase in the number of revertants was observed

PRE-INCUBATION ASSAY
- Precipitation: present at the start of the incubation period, at concentration 5000 μg/plate
- No precipitate was observed at the end of the incubation period
- Mutagenicity: no increase in the number of revertants was observed

Cytotoxicity:
TA 1535, TA1537, TA98, TA100: No cytotoxicity up to and including 5000 μg/plate with and without S9
WP2uvrA: cytotoxicity at 5000 μg/plate with and without S9 in the direct plate assay only

Applicant's summary and conclusion

Conclusions:
Based on the outcome of the AMES test, it is concluded that SDH is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

An AMES test was performed according to OECD guideline and in accordance with GLP principles. All bacterial strains showed negative responses up to 5000 µg /plate, i.e. no significant dose-related increase in the number of revertants with or without metabolic activation was seen. No cytotoxicity was observed up to including 5000 µg/plate with the exception of WP2uvrA at the highest test concentration. 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.

Based on the results of this study it is concluded that SDH is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay with or without metabolic activation.