[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 April 2017 - 18 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

batch number: 616F-2072 (NBK-003072-796)

Method

Target gene:

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100: histidine gene
Escherichia coli WP2uvrA strain: tryptophan gene

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 17, 52, 164, 512, 1600 and 5000 µg/plate (+/- S9 mix)
The dose-range finding test was performed with the strains TA100 and the WP2uvrA, both with and without S9-mix, using the following concentrations: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate

Vehicle / solvent:

Ethanol. The stock solution was completely dissolved after sonication. Test item concentrations were used within 2 hours after preparation

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

Controls 2

Negative solvent / vehicle controls:

yes

Remarks:

Saline

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

sodium azide

Remarks:

For TA1535 strain, without metabolic activation, at concentration of 5 µg, in both direct plate assay and pre-incubation assay

Controls 3

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

other: ICR-191 (Sigma)

Remarks:

For TA1537 strain, without metabolic activation, at concentration of 2.5 µg (used only in direct plate assay, not in pre-incubation assay)

Controls 4

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

2-nitrofluorene

Remarks:

For TA1537 strain, without metabolic activ., at conc. of 15 µg, only in the preincubation assay and not in Direct plate assay. For TA98 strain, without metabolic activ., at conc. 10 micrograms, in both direct plate assay and in preincubation assay

Controls 5

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

methylmethanesulfonate

Remarks:

For TA100 strain, without metabolic activation, at concentration of 650 µg, in both direct plate assay and pre-incubation assay.

Controls 6

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

For WP2uvrA strain, without metaboilic activation, at concentration of 10 µg, in both direct plate assay and pre-incubation assay

Controls 7

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-aminoanthracene (Sigma)

Remarks:

For TA1535, TA1537, TA98, TA100, WP2uvrA, with metabolic activ, in both direct plate and preincubation assay. Conc: TA1535 and TA1537= 2.5 µg; TA98 = 1 µg, TA100 = 1 µg in direct plate and 5 µg in preincubation ; WP2uvrA = 15 µg

Controls 8

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

2-nitrofluorene

Remarks:

For TA98 strain, without metabolic activation, at concentration of 10 µg in both direct plate assay and preincubation assay

Details on test system and experimental conditions:

METHOD OF APPLICATION:
in agar (plate incorporation); test concentrations were prepared immediately before use.

NUMBER OF REPLICATIONS:
triplicate

DETERMINATION OF CYTOTOXICITY
bacterial background lawn

EXPOSURE DURATION
48 +/- 4 h

Evaluation criteria:

A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following 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.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

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 (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) 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 (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 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 results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

At the end of the incubation period, the test item precipitated from the concentration of 512 µg/plate in the absence of S9-mix, from 1600 µg/plate in the presence of S9-mix, and heavily at 5000 µg/plate with S9 mix.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Since the test item heavily precipitated at concentration of 5000 µg/plate without S9-mix, the cytotoxicity at this dose could not be determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

At the end of the incubation period, the test item precipitated from the concentration of 512 µg/plate in the absence of S9-mix, from 1600 µg/plate in the presence of S9-mix, and heavily at 5000 µg/plate with S9 mix.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Since the test item precipitated heavily at the concentration of 5000 microgr/plate without S9-mix, at this dose level cytotoxicity could not be determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

At the end of the incubation period, the test item precipitated from the concentration of 512 µg/plate in the absence of S9-mix, from 1600 µg/plate in the presence of S9-mix, and heavily at 5000 µg/plate with S9 mix.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Since the test item heavily precipitated at concentration of 5000 µg/plate, cytotoxicity at this concentration could not be determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

At the end of the incubation period, the test item precipitated from the concentration of 1600 µg/plate in the absence of S9-mix, and heavily at 5000 µg/plate with S9 mix.

Cytotoxicity / choice of top concentrations:

other: Cytotoxicity was not observed in presence of S9-mix; cytotoxicity was observed in absence of S9-mix. Since the test item precipitated heavily at concentration of 5000 µg/plate in presence of S9-mix, cytotoxicity at this dose could not be determined

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

At the end of the incubation period, the test item heavily precipitated at the concentration of 5000 µg/plate in presence and absence of S9 mix.

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

No cytotoxicity was observed up to the dose level of 1600 µg/plate. Since the test item heavily precipitated at the concentration of 5000 µg/plate in presence and absence of S9 mix, cytotoxicity could not be determined at this dose level

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Test item precipitated at the start of the incubation period at the concentration of 5000 µg/plate and at 1600 and 5000 µg/plate at the end of the incubation period

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Since the test item heavily precipitated at the concentration of 5000 µg/plate in presence and absence of S9 mix, cytotoxicity could not be determined at this dose level.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Test item precipitated at the start of the incubation period at the concentration of 5000 µg/plate, and at 1600 and 5000 µg/plate at the end of the incubation period

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Since the test item heavily precipitated at the concentration of 5000 µg/plate in presence and absence of S9 mix, cytotoxicity could not be determined at this dose level.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Test item precipitated at the start of the incubation period at the concentration of 5000 µg/plate, and at 1600 and 5000 µg/plate at the end of the incubation period

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Since the test item heavily precipitated at the concentration of 5000 µg/plate in presence and absence of S9 mix, cytotoxicity could not be determined at this dose level.

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 9

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Precipitation at start of incubation at 5000 µg/plate, at end incubation at 1600 and 5000 µg/plate. At 17 and 512 µg/plate with S9-mix up to 4fold increases vs solv control, due to low solv control values. Increases within historical control data range.

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Since the test item heavily precipitated at the concentration of 5000 µg/plate in presence and absence of S9 mix, cytotoxicity could not be determined at this dose level

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 10

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Test item precipitated at the start of the incubation period at the concentration of 5000 µg/plate, and at 1600 and 5000 µg/plate at the end of the incubation period

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Since the test item heavily precipitated at the concentration of 5000 µg/plate in presence and absence of S9 mix, cytotoxicity could not be determined at this dose level.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

DOSE RANGE finding study was performed with the strains TA100 and WP2 uvrA, both with and without S9-mix, at the concentration of: 1.7; 5.4; 17; 52; 164; 512; 1600; 5000"µg/plate . Those were tested in triplicate. The results are shown in the "test results" table, above, under teh remark "direct plate assay".

Remarks on result:

other: This result from Pre-incubation assay

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative.

In an Ames test, the test substance showed to be negative with and without metabolic activation in 5 strains: TA1535, TA1537, TA98, TA100 and WP2 uvrA

Executive summary:

The test substance was tested in the Bacterial Reverse Mutation Test with histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100), and with a tryptohan-requiring strain of Escherichia coli (WP2uvrA) in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

The study was performed according to OECD guideline 471. Six concentrations (17, 52, 164, 512, 1600 and 5000 µg/plate) were tested in triplicate, each assay was conducted twice (direct mutagenicity plate test and pre-incubation mutation test).

In the direct mutagenicity plate test, the test item precipitated on the plates at dose levels of 1600 and 5000 μg/plate. In the pre-incubation mutation experiment, the test item precipitated with all the strains at dose levels of 5000 µg/plate in presence of the metabolic activation system, and in the plates containing WP2uvrA at dose levels of 5000 µg/plate in absence of metabolic activation system.

In the plates with the precipitate, neither the bacterial background lawn nor the number of revertants of this dose level could be determined.

In the other concentration ranges, the test item did not induce a significant dose-related increase in the number of revertant (His⁺) colonies of Salmonella typhimuriumand in the number of revertant (Trp⁺) colonies of Escherichia coli, both in the absence and presence of S9-metabolic activation. 

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.