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 November 2017 - 20 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 July 1997
Deviations:
no
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Physical appearance: dark amber solid
- Storage conditions: at room temperature

Method

Target gene:
Salmonella typhimurium: histidine gene
Escherichia coli: tryprophan gene
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) from rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Test concentrations with justification for top dose:
Dose range finding test (TA100 and WP2uvrA, with and without S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate (reported as part of the first experiment)
First experiment (TA1535, TA 1537 and TA98): without and with S9: 31, 63, 125, 250, 500 and 1000 μg/plate
First additional experiment (TA1535, TA 1537 and TA98): without and with S9: 0.63, 1.25, 2.5, 5, 10 and 20 μg/plate
Second experiment (all strains) without and with S9: 3.1, 6.3, 12.5, 25, 50, 100 and 150 μg/plate
Second additional experiment (TA1535, TA1537, TA98 and TA100) without S9: 0.31, 0.63, 1.25, 2.5, 5 and 10 μg/plate
Vehicle / solvent:
- Solvent used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: the solvent is according to OECD guideline 471 and the test substance dissolved in DMSO (based on visual assessment).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191; 2-aminoanthracene
Remarks:
See table 1 for more details on postive control substances
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 +/- 4 hours

NUMBER OF REPLICATIONS: 3

PERFORMANCE OF THE ASSAY: The following solutions were successively added to 3 ml molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 mL of a dilution of the test item in DMSO and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C

CYTOTOXICITY:
- Cytotoxicity was examined by the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies

COLONY COUNTING:
The revertant colonies 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:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle 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 the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up 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.

In addition to the criteria stated above, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

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 this laboratory.
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.

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:
cytotoxicity
Remarks:
In experiment 1: at and above 31 µg/plate (without S9); at and above 125 µg/plate (with S9); in experiment 2: at and above 3.1 µg/plate (without S9); at and above 100 µg/plate (with S9); in experiment 2A: at 10 µg/plate (without S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In experiment 1: at and above 31 µg/plate (without S9); at and above 63 µg/plate (with S9); in experiment 2: at and above 3.1 µg/plate (without S9); at and above 50 µg/plate (with S9); in experiment 2A: at and above 5 µg/plate (without S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In experiment 1: at and above 63 µg/plate (without S9); at and above 125 µg/plate (with S9); in experiment 2: at and above 12.5 µg/plate (without S9); at and above 50 µg/plate (with S9); in experiment 2A: at 10 µg/plate (without S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In experiment 1: at and above 17 µg/plate (without S9); at and above 17 µg/plate (with S9); in experiment 2: at and above 6.3 µg/plate (without S9); at and above 50 µg/plate (with S9); in experiment 2A: at and above 5 µg/plate (without S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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:
In experiment 1: at and above 164 µg/plate (without S9); at and above 164 µg/plate (with S9); in experiment 2: at and above 100 µg/plate (without S9); at 150 µg/plate (with S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Due to severe cytotoxicity in both the first and the second experiment, these experiments were repeated. In the first additional experiment (1A) the test item was tested in the strains TA1535, TA1537 and TA98 in the presence and the absence of S9. In the second additional experiment (2A) the test iten was tested in the strains TA1535, TA1537, TA98 and TA100 in the absence of S9.

PRECIPITATION:
- Dose-range finding/first experiment: at the start of the incubation period at concentrations of 1600 µg/plate and upwards and at 5000 µg/plate at the end of the incubation period except for tester strain TA100 in the absence of S9-mix where precipitate was observed at dose level of 1600 μg/plate and upwards in the dose-range finding test.
- First additional experiment: no precipitation observed
- Second experiment: no precipitation observed
- Second additional experiment: at the start of the incubation period at the concentration of 10 µg/plate and no precipitate was observed at the end of the incubation period.

CYTOTOXICITY
- Dose-range finding/first experiment: Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the presence and in the absence of S9.
- First additional experiment: no cytotoxicity observed.
- Second experiment: observed in all tester strains in the presence and in the absence of S9.
- Second additional experiment: observed at the highest dose level tested in all tester strains except for tester strain TA1537 and TA100 where toxicity was observed at dose levels of 5 µg/plate and upwards.

MUTAGENICITY
- Dose-range finding/first experiment: no
- First additional experiment: no
- Second experiment: no
- Second additional experiment: no

HISTORICAL CONTROL DATA
- The negative control values and the strain-specific positive control values were within the laboratory historical control data ranges.

Applicant's summary and conclusion

Conclusions:
The results of an Ames test, performed according to OECD guideline 471 and GLP principles, showed that INCA 460: DEPDA Adduct is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.