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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013-10-16 to 2013-11-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study is conducted in compliance with GLP regulations and OECD/EU guidelines.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

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:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder

Method

Target gene:
histidine and tryptophan mutation
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:
S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
Test concentrations with justification for top dose:
- Salmonella typhimurium TA98, TA100, TA1535 and TA1537: ±S9 Mix: 1581; 500; 158; 50; 15.8 and 5 μg/plate
- Escherichia coli WP2 uvrA: ±S9 Mix: 5000; 1581; 500; 158; 50 and 15.8 μg/plate
- Additional Confirmatory Experiment: Salmonella typhimurium TA100: -S9 Mix: 1581, 500, 158, 50, 15.8 and 5 μg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: The vehicle was compatible with the survival of the bacteria and the S9 activity and was chosen based on the results of the preliminary Solubility Test.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Test medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylenediamine (NPD)
Remarks:
Salmonella TA98, w/o metabolic activation
Untreated negative controls:
yes
Remarks:
Test medium
Negative solvent / vehicle controls:
yes
Remarks:
Ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Salmonella TA100 and TA1535, w/o metabolic activation
Untreated negative controls:
yes
Remarks:
Test medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Salmonella TA1537, w/o metabolic activation
Untreated negative controls:
yes
Remarks:
Test medium
Negative solvent / vehicle controls:
yes
Remarks:
Ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
All Salmonella strains, w metabolic activation
Untreated negative controls:
yes
Remarks:
Test medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
E.coli, w metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation), preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: at least 48 h

SELECTION AGENT (mutation assays): histidine-biotin (0.5mM) and tryptophan (2 mg/mL)

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
The colony numbers on the control, positive control and the test plates were determined, the mean values, standard deviations and the mutation rates were calculated.
Mutation Rate = Mean revertants at the test item (or control) treatments/Mean revertants of vehicle control

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.
According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.
Criteria for a Negative Response:
A test article is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
not applicable

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Precipitation: Precipitate was observed at the concentrations of the 5000 and 1581 μg/plate in the absence and also in the presence of exogenous metabolic activation (±S9 Mix) and at 500 μg/plate, in absence of metabolic activation (-S9 Mix).

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with Salmonella typhimurium TA98 and TA100 strains in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.
In the Informatory Toxicity Test the concentrations examined were: 5000, 1581, 500, 158, 50, 15.8 and 5 μg/plate.
Strong cytotoxic effects of the test item were noticed in both examined S. typhimurium strains. Absence of revertant growth and reduced or slightly reduced background lawn development was observed in S. typhimurium TA98 at 5000 μg/plate, without and with addition of metabolic activation (±S9 Mix), at 1581 μg/plate, without metabolic activation (-S9 Mix) and in S. typhimurium TA100 at 5000 and 1581 μg/plate (±S9 Mix).
Revertant colony numbers below the vehicle and corresponding historical control data ranges, and affected background lawn development (slightly reduced background lawn development) was observed in S. typhimurium TA98 at 1581 μg/plate (+S9 Mix).
Indicating the inhibitory effect of the test item, the revertant colony numbers were lower than the revertant colony numbers of the vehicle control and were below the historical control data range at 500 μg/plate in S. typhimurium TA98, without metabolic activation (-S9 Mix) and in S. typhimurium TA100, without and with addition of metabolic activation (±S9 Mix).
The revertant colony numbers remained in the vehicle control data range, however were below the corresponding historical control data range in S. typhimurium TA98 at 15.8 μg/plate (-S9 Mix) and at 500 μg/plate (+S9 Mix); furthermore in S. typhimurium TA100 at 158 μg/plate (-S9 Mix). This change was evaluated rather as biological variability of the applied test system than a real test item effect.
In the Informatory Toxicity Test the observed revertant colony number increases were of minor intensity and far below the biologically relevant thresholds for being positive. Higher revertant colony counts within the corresponding historical control data ranges were obtained at 50 μg/plate in S. typhimurium TA98 and in S. typhimurium TA100 (+S9 Mix).
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Information on Results, Cytotoxicity and Historical Control Data

Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of DOCU Red 116 in four independent experiments, in a plate incorporation test (Experiment I, Initial Mutation Test) and in pre-incubation tests (Experiment II, Confirmatory Mutation Test and Additional and Second Additional Confirmatory Mutation Tests). The Initial and Confirmatory Mutation Tests were conducted with and without metabolic activation (S9 Mix). In the Additional Confirmatory Mutation Test the Salmonella typhimurium TA1535, TA1537 strains and in the Second Additional Confirmatory Mutation Test the Salmonella typhimurium TA100 strain were investigated in the absence of metabolic activation (-S9 Mix).

The concentrations, including the controls, were tested in triplicate (positive and negative controls were run concurrently).

Following concentrations of DOCU Red 116 were tested in the performed experiments (Plate Incorporation Test and Pre-Incubation Tests) and were chosen based on results obtained in the Informatory Toxicity Test: in Salmonella typhimurium TA98, TA100, TA1535 and TA1537: ±S9 Mix: 1581; 500; 158; 50; 15.8 and 5 μg/plate;

in Escherichia coli WP2 uvrA: ±S9 Mix: 5000; 1581; 500; 158; 50 and 15.8 μg/plate.

In the Initial Mutation Test the obtained revertant colony numbers were significantly higher than the revertant colony numbers of the vehicle control and these higher counts were above the historical control data range in S. typhimurium TA1535 at 15.8 μg/plate with addition of metabolic activation (+S9 Mix). This value was unique, not accompanied with unequivocal dose relationship and remained below the threshold for being positive. Therefore this value was not evaluated as a real test item effect.

In the Initial Mutation Test the further observed higher revertant colony numbers (higher than the revertant colony numbers of the vehicle control plates) remained within the corresponding historical control data ranges. Higher revertant colony numbers were observed at 158 μg/plate in the case of S. typhimurium TA100 (±S9 Mix), in E. coli WP2 uvrA with addition of metabolic activation (+S9 Mix); at 50 μg/plate in TA98 (-S9 Mix), in TA100 and in TA1535 (±S9 Mix) and in E. coli WP2 uvrA (+S9 Mix); at 15.8 μg/plate in TA100 (±S9 Mix), in TA1535 (-S9 Mix) and in E. coli WP2 uvrA (+S9 Mix); and at 5 μg/plate in TA98 and in TA100 with addition of metabolic activation (+S9 Mix). The higher revertant colony counts remained far below the threshold for being positive in all cases.

The obtained revertant colony numbers remained in the actual vehicle control data range, however were above the corresponding historical control data range (without any biological significance) in E. coli WP2 uvrA at 50 μg/plate (-S9 Mix).

In the Initial Mutation Test the inhibitory effect of the test item was indicated by decreased revertant colony counts and affected colony and background lawn development.

Revertant colonies were not noticed on the plates; furthermore the background lawn development was affected (reduced or slightly reduced background lawn development) at the highest concentration of 1581 μg/plate in S. typhimurium TA100 and TA1535 without and with addition of metabolic activation (±S9 Mix); furthermore in TA1535 at 500 μg/plate (-S9 Mix).

Reduced or slightly reduced background lawn development and colony numbers below the vehicle and historical control data ranges were noted at 5000 μg/plate in E. coli WP2 uvrA (±S9 Mix); at 1581 μg/plate in S. typhimurium TA98 (±S9 Mix) and TA1537 (-S9 Mix); and at 500 μg/plate in TA1535 (+S9 Mix) and TA1537 (-S9 Mix).

The lower revertant colony numbers (compared to the revertant colony numbers of the vehicle control) remained in the corresponding historical control data ranges, however reduced background lawn development was indicative for the inhibitory effect of the test item at 1581 and 500 μg/plate in S. typhimurium TA1537, with addition of metabolic activation (+S9 Mix).

Indicating the inhibitory effect of the test item the lower (in comparison with the revertant colony numbers of the vehicle control) revertant colony numbers were below the historical control data range (but the background lawn development was not affected) at 500 μg/plate in S. typhimurium TA98 (-S9 Mix) and in TA100 (±S9 Mix).

The obtained lower revertant colony numbers (compared to the revertant colony numbers of the vehicle control plates) remained in the corresponding historical control data ranges at 1581 μg/plate in in E. coli WP2 uvrA (±S9 Mix); at 500 μg/plate in E. coli WP2 uvrA (-S9 Mix); at 158 μg/plate in S. typhimurium TA1535 (-S9 Mix), in TA1537 (±S9 Mix); at 50 μg/plate in TA1537 (-S9 Mix) and at 5 μg/plate in TA1535 (-S9 Mix).

The test item concentrations of DOCU Red 116 tested in Experiment II were the same as investigated in the Experiment I.

In this experimental part an increase in revertant colony numbers was obtained in S. typhimurium TA100 at a concentration level of 15.8 μg/plate without metabolic activation (-S9 Mix). This increase was biologically relevant and above the threshold for being positive (the obtained revertant colony counts were more than twice as high as the revertant colony number of the corresponding vehicle control). This increase was unique, remained within the historical control data range and was not attained with unequivocal dose-relationship. Therefore it was considered rather equivocal than a real test item effect. Because of the marginal results, this part (S. typhimurium TA100, in absence of metabolic activation) of the Confirmatory Mutation Test was repeated in the Second Additional Confirmatory Mutation Test (discussed below).

In the Confirmatory Mutation Test further higher revertant colony numbers were noticed in Escherichia coli WP2 uvrA at 50 and 15.8 μg/plate without metabolic activation. These revertant colony number increases were above the historical control data range, however remained far below the genotoxicological threshold for being positive. Therefore they were not evaluated as a test item effect.

In the Confirmatory Mutation Test the revertant colony numbers were slightly higher than the revertant colony numbers of the vehicle control plates at 158 μg/plate in Escherichia coli WP2 uvrA (-S9 Mix), at 50 μg/plate in S. typhimurium TA100 (-S9 Mix), in TA1535 (+S9 Mix); at 15.8 μg/plate in TA1535 and in TA1537 (+S9 Mix) and at 5 μg/plate in TA100 (-S9 Mix), in TA1535 (+S9 Mix). All of these increases occurred sporadically and remained in the corresponding historical control data ranges.

In the Confirmatory Mutation Test the inhibitory effect of the test item was indicated with lower or absent revertant colony counts (compared to the vehicle control) mostly below the corresponding historical control data ranges and reduced or slightly reduced background lawn development.

As inhibitory effect of the test item, absence of revertant growth and reduced background lawn development was obtained in S. typhimurium TA1535 at 1581 μg/plate (-S9 Mix), in TA1537 at 1581 μg/plate (±S9 Mix); furthermore in TA1537 at 500 and 158 μg/plate (-S9 Mix).

Low revertant colony numbers (below the corresponding historical control data ranges) and reduced or slightly reduced background lawn development was obtained at 5000 μg/plate in Escherichia coli WP2 uvrA (±S9 Mix), at 1581 μg/plate in Escherichia coli WP2 uvrA (-S9 Mix), in S. typhimurium TA98 and TA100 (±S9 Mix); at 500 μg/plate in TA98 (-S9 Mix), in TA100 (±S9 Mix), in TA1537 (+S9 Mix); at 158 μg/plate in TA98 (-S9 Mix).

The lower revertant colony numbers were below the historical control data ranges however the background lawn development was not affected in S. typhimurium TA100 at 158 μg/plate (-S9 Mix).

The lower revertant colony numbers remained within the historical control data ranges, but additional reduced or slightly reduced background lawn development indicate an unequivocal inhibition in S. typhimurium TA98 at 500 μg/plate (+S9 Mix); in TA1535 at the concentrations of 500 and 158 μg/plate (-S9 Mix) and in the concentration range of 1581-158 μg/plate (+S9 Mix), in TA1537 at 50 μg/plate (-S9 Mix) and at 158 μg/plate (+S9 Mix) and in Escherichia coli WP2 uvrA at 1581 μg/plate (+S9 Mix).

As sign of the inhibition, the revertant colony numbers (within the historical control data ranges) were significantly lower than the revertant colony numbers of the vehicle control in S. typhimurium TA1535 at 50 and 15.8 μg/plate (-S9 Mix), and in TA1537 at 15.8 μg/plate (-S9 Mix).

Further lower revertant colony counts were obtained without any biological significance in the S. typhimurium TA98 at 50 μg/plate (+S9 Mix), in TA1535 and in TA1537 at 5 μg/plate (-S9 Mix); furthermore in Escherichia coli WP2 uvrA, at 500 μg/plate, with and without metabolic activation (±S9 Mix).

Because of the obtained test item inhibitory effect in the Confirmatory Mutation Test, complementation of the examined concentration levels was considered as necessary in the case of Salmonella typhimurium TA1535 and TA1537 strains in the absence of metabolic activation (-S9 Mix). At these strains and experimental parts the validity criterion regarding the number of analyzable concentrations (at least five) and the number of evaluated non-toxic dose levels (at least three) was not fulfilled; therefore the following, modified concentration range was additionally examined in an Additional Confirmatory Mutation Test: 50; 15.8; 5; 1.6; 0.5; 0.16 μg/plate.

In this additional test the obtained results were similar (within the biological variability range of the applied test system) to the results obtained in the Confirmatory Mutation Test in the case of Salmonella typhimurium TA1537 at the common concentrations of 50, 15.8 and 5 μg/plate.

Revertant colonies were not observed and the background lawn development was also affected (reduced background lawn development)was obtained at 50 μg/plate, the significantly lower revertant growth remained within the historical control data range and slightly reduced background lawn development confirmed the strong test item effect at 15.8 μg/plate. The slightly decreased revertant colony numbers in Salmonella typhimurium TA1537 at 1.6 and 0.5 μg/plate were evaluated as reflecting the biological variability of the applied test system.

In the Additional Confirmatory Mutation Test the lower revertant colony numbers (lower than the revertant colony numbers of the vehicle control) remained within the historical control data range but slightly reduced background lawn development confirmed unequivocally the inhibition in Salmonella typhimurium TA1535, at 50 μg/plate.

At this strain revertant colony number increases were obtained at the lower concentration levels. These increases were above the corresponding historical control data range at 15.8 and at 5 μg/plate. These increases were far below the genotoxicological threshold for being positive and especially in view of the results of the Confirmatory Mutation Test these increases remained within the biological variability of the test system. Further, increases were obtained within the biological variability range of the applied test system at 1.6, 0.5 and 0.16 μg/plate.

In the Second Additional Confirmatory Mutation Test the Salmonella typhimurium TA100 strain was examined with the same concentration levels as already applied in the Initial and Confirmatory Mutation Tests. The obtained revertant colony number increases at 50, 15.8 and at 5 μg/plate remained in the corresponding historical control data range and remained below the threshold for being positive. Similarly to the Confirmatory Mutation Test results inhibitory effect of the test item (decreased revertant colony numbers below the historical control data range, and reduced or slightly reduced background lawn development) was observed down to and including the concentration level of 158 μg/plate.

The revertant colony numbers of the untreated and ultrapure water control plates in the different experimental phases were slightly higher or lower than the DMSO control plates.

The higher or lower revertant counts of these controls remained mostly in the corresponding historical control data ranges. In the Second Additional Confirmatory Mutation Test in the case of Salmonella typhimurium TA100 the revertant colony count of the water control plates was below the corresponding historical control data range, without metabolic activation (-S9 Mix).

The investigated reference mutagens (positive controls) showed the expected (at least 3-fold) increase in induced revertant colonies over the mean value of the respective vehicle control and were in the corresponding historical control data ranges in all cases.

In the tests revertant colony number increases above the corresponding historical control data ranges were noticed; however these increases were sporadic, unique values, were not attained by dose-relationship, therefore were considered rather as biological variability of the applied test system and not a real test item effect. The marginal, equivocal positive results of the Confirmatory Mutation Test in Salmonella typhimurium TA100 in absence of metabolic activation was not confirmed by the Second Additional Confirmatory Mutation Test.

Following the plate incorporation procedure precipitate was noticed at 5000, 1581, and 500 μg/plate in absence (-S9 Mix) and at 5000 and 1581 μg/plate in the presence of metabolic activation (+S9 Mix); following the pre-incubation procedure precipitate was noticed at 5000 and 1581 μg/plate, in absence and also in presence of metabolic activation (±S9 Mix).

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The reported data of the mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, DOCU Red 116 is considered non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

Five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of DOCU Red 116 in independent experiments, in a plate incorporation test (Experiment I, Initial Mutation Test), and in a pre-incubation tests (Experiment II, Confirmatory Mutation Test and additional, partial confirmatory tests). The Initial and Confirmatory Mutation Tests were conducted with and without metabolic activation (S9 Mix).

The test item was dissolved (suspended) in dimethyl sulfoxide (DMSO). In the Initial and Confirmatory Mutation Tests the tested concentrations were:

in Salmonella typhimurium TA98, TA100, TA1535 and TA1537: ±S9 Mix: 1581; 500; 158; 50; 15.8 and 5 μg/plate;

in Escherichia coli WP2 uvrA: ±S9 Mix: 5000; 1581; 500; 158; 50 and 15.8 μg/plate.

Because of the observed test item effects in the Confirmatory Mutation Test, additional confirmatory experiments (partial tests) were carried out using Salmonella typhimurium TA1535, TA1537 strains (Additional Confirmatory Mutation Test) with the concentrations of: 50, 15.8, 5, 1.6, 0.5 and 0.16 μg/plate and using Salmonella typhimurium TA100 strain (Second Additional Confirmatory Mutation Test) with the concentrations of 1581; 500; 158; 50; 15.8 and 5 μg/plate. The additional confirmatory tests were performed in absence of exogenous metabolic activation.

The test item concentrations, including the controls, were tested in triplicate.

In the performed experiments positive and negative (vehicle) controls run concurrently. The revertant colony numbers of vehicle control plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants in the vehicle controls (were within the corresponding historical control data ranges). The reference mutagens showed a distinct increase of induced revertant colonies.

In the performed experimental phases (in the Confirmatory Mutation Test together with the Additional Confirmatory Mutation Tests) were at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain. The validity criteria of the study were fulfilled.

In the tests revertant colony number increases above the corresponding historical control data ranges were noticed; however these increases were sporadic, unique values and were not attained by dose-relationship. Therefore they were considered rather as biological variability of the applied test system and not as a real test item effect. The marginal, equivocal positive results of the Confirmatory Mutation Test in Salmonella typhimurium TA100 in absence of metabolic activation was not confirmed by the Second Additional Confirmatory Mutation Assay.

Inhibitory effect of the test item was observed in all examined strains. The inhibition appeared unequivocally down to and including the concentration of 15.8 μg/plate (Salmonella typhimurium TA1537, Confirmatory Mutation Test, Additional Confirmatory Mutation Test) in absence; and down to 158 μg/plate (Salmonella typhimurium TA1535 and TA1537, Confirmatory Mutation Test) in presence of exogenous metabolic activation.

The reported data of the mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, DOCU Red 116 is considered non-mutagenic in this bacterial reverse mutation assay.