Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 Apr to 10 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21, 1997
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Zschimmer and Schwarz GmbH + Co. KG.
- Identification: 1739 MIPA-CITRAT
- Batch: 207459-01
- Purity/Composition: UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Test item storage: At room temperature
- Stable under storage conditions until: 13 January 2018 (expiry date)
- Solubility in vehicle: Water: Not indicated; Dimethyl sulfoxide: Not indicated
- Stability in vehicle: Water: Stable; Dimethyl sulfoxide: Not indicated

OTHER SPECIFICS
- Appearance: Clear yellow liquid
- Purity/Composition correction factor: Yes, according to the purity: 1.49

Method

Target gene:
- S. typhimurium: his
- E. coli: trp
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 from Aroclor 1254 (i.p.) induced rats livers
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from Aroclor 1254 (i.p.) induced rats livers
Test concentrations with justification for top dose:
DOSE RANGE FINDING TEST
Strains TA100 and the WP2uvrA, with and without S9-mix: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate

EXPERIMENT 1 (DIRECT PLATE ASSAY)
- Strains TA98, TA1535 and TA1537, with and without S9-mix: 52, 164, 512, 1600, 5000 μg/plate

EXPERIMENT 2 (PRE-INCUBATION ASSAY)
- Strains TA98, TA100, TA1535, TA1537 and WP2 uvrA, with and without S9-mix: 52, 164, 512, 1600, 5000 μg/plate
Vehicle:
DOSE RANGE FINDING TEST and EXPERIMENT 1 and 2
- Vehicle used: Milli-Q water

REPEAT EXPERIMENT 2
- Vehicle used: dimethyl sulfoxide

JUSTIFICATION
The use of two solvents has no effect on the study integrity.
Controls
Negative controls:
no
Solvent controls:
yes
Remarks:
Milli-Q water or dimethyl sulfoxide
True negative controls:
no
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
- DOSE-RANGE FINDING TEST and EXPERIMENT 1: in agar (plate incorporation)
- EXPERIMENT 2: preincubation

DURATION
- Preincubation period (EXPERIMENT 2 only): 30 minutes by 70 rpm at 37°C
- Exposure duration: 48 ± 4 h in the dark at 37.0 ± 1.0°C

NUMBER OF REPLICATIONS
3 plates/dose

COLONY COUNTING
Revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.
Evaluation criteria:
see section "Any other information on materials and methods incl. tables"

Results and discussion

Test resultsopen allclose all
Species / strain:
other: TA1535, TA1537, TA100 and TA98
Remarks:
Dose range Experiment and Experiment 1: Direct Plate Assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Species / strain:
E. coli WP2 uvr A
Remarks:
Dose range Experiment: Direct Plate Assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98
Remarks:
Experiment 2: Pre-Incubation Assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
Experiment 2: Pre-Incubation Assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Experiment 2: Pre-Incubation Assay
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity:
yes
Remarks:
Cytotoxicity, as evidenced by a reduction in the bacterial background lawn, was only observed in tester strain TA100 in the presence of S9-mix at the highest tested concentration.
Vehicle controls valid:
yes
Negative controls valid:
not applicable
Positive controls valid:
yes
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation, Direct Plate Assay: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.
- Precipitation, Pre-Incubation Assay: Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period.

Applicant's summary and conclusion