Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 July - 01 August, 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:
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:
liquid: viscous
Details on test material:
- Appearance: Brown viscous liquid
- Storage condition of test material: At room temperature

Method

Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan 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 S9-mix induced by Aroclor 1254 (500 mg/kg body weight)
Test concentrations with justification for top dose:
- Dose-range finding (TA100 and WP2 uvrA, with and without S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (reported as part of the mutation experiment)
- Mutation experiment (TA1535, TA1537 and TA98, with and without S9): 17, 52, 164, 512, 1600 and 5000 μg/plate
- Test item concentrations were used within 1 hour after preparation
Vehicle / solvent:
- Solvent used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent: the test item dissolved visually in DMSO, which is a solvent recommended by international guidelines.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See the section 'any other information on materials and methods'
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

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

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION: Top agar in top agar tubes was melted by heating to 45°C. Fresh bacterial culture, dilution of the test item in DMSO and either S9-mix or 0.1 M phosphate buffer were added to the molten top agar. The ingredients were mixed on a vortex and the content of the top agar tube was poured onto a selective agar plate.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
DECISION 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 vehicle control, and the total number of revertants in tester strain TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.

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 vehicle control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three times the concurrent vehicle control.

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 the test facility.
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
Remarks:
(based on a single experiment)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
(based on a single experiment)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
(based on a single experiment)
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
DOSE-RANGE FINDING
- Precipitation: Not observed
- Cytotoxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 in the absence and presence of S9-mix at the highest test concentration (5000 μg/ plate). In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested (when compared with the historical control data).
- Mutagenicity:
-- In tester strain TA100, the test item induced up to 2.7- and 2.4-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were above the laboratory historical control data ranges and more than two-fold the concurrent solvent control.
-- In tester strain WP2uvrA, the test item induced up to 5.8- and 6.3-fold dose-related increases in the number of revertant colonies in the absence and presence of S9-mix, respectively. The increases observed were above the laboratory historical control data ranges and more than two-fold the concurrent solvent control.

MUTATION EXPERIMENT
- Precipitation: Not observed
- Cytotoxicity: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in all three tester strains in the absence and presence of S9-mix.
- Mutagenicity: No increase in the number of revertants was observed upon treatment with the test item.

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.

- Positive historical control data:

TA1535 TA1537 TA98
S9-mix - + - + - +
Range 128 – 1530 73 – 1206 58 – 1407 54 – 1051 365 – 1978 250 – 1977
Mean 901 239 817 340 1355 903
SD 174 115 354 160 230 357
n 2400 2296 2051 2337 2357 2367

TA100 WP2uvrA
S9-mix - + - +
Range 439 – 1993 408 - 2379 93 – 1958 111 - 1359
Mean 905 1249 1059 444
SD 163 371 506 144
n 2402 2354 2153 2232

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between May 2016 and May 2018.

- Negative (solvent/vehicle) historical control data:

TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 3 – 29 3 – 27 3 – 20 3 – 23 8 - 41 8 – 55 61 – 176 60 - 160 10 – 59 9 - 67
Mean 10 11 6 6 16 22 110 106 26 33
SD 3 4 2 3 5 7 17 20 6 8
n 2458 2426 2402 2352 2416 2458 2473 2398 2237 2217

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between May 2016 and May 2018.

Applicant's summary and conclusion

Conclusions:
In an AMES test, performed according to OECD 471 and GLP, the substance was found to be mutagenic in the tester strains TA100 of the Salmonella typhimurium reverse mutation assay and WP2uvrA of the Escherichia coli reverse mutation assay, both in absence and presence of metabolic activation.
Executive summary:

The mutagenic potential of the substance and/or its metabolites was assessed in an Ames test, performed according to OECD guideline 471 and GLP principles. The test item was tested in several strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and in one strain of Escherichia coli (WP2uvrA) in the absence and in the presence of a metabolic activation system (S9-mix). Concentrations up to and including 5000 µg/plate were used in a dose-range finding test (tester strains TA100 and WP2uvrA) and a mutation assay (tester strains TA1535, TA1537 and TA98), in absence and presence of S9 -mix.

The test item did not precipitate on the plates. Cytotoxicity was seen in all Salmonella typhimurium strains at 5000 µg/plate, both in presence and absence of metabolic activation. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

In the absence of S9-mix, the test item induced up to 2.7- and 5.8-fold increases in the number of revertant colonies compared to the solvent control in the tester strains TA100 and WP2uvrA, respectively. In the presence of S9-mix, the test item induced up to 2.4- and 6.3-fold increases in the number of revertant colonies compared to the solvent control in the tester strains TA100 and WP2uvrA, respectively. Since the increases observed were above the laboratory historical control data range, more than two-fold and were dose-related, these increases are considered to be biologically relevant.

Since the test results of the mutation experiment showed clear positive responses, with dose-dependency, a follow-up experiment was not performed. The results of the solvent control and the positive controls were within the historical control data ranges of the test facility.

In conclusion, based on the results of this study it is concluded that the substance is mutagenic in the tester strains TA100 of the Salmonella typhimurium reverse mutation assay and WP2uvrA of the Escherichia coli reverse mutation assay, both in absence and presence of metabolic activation.