Registration Dossier

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:
2013-03-20 to 2013-04-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to OECD/ EU guideline.

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:
other: colourless liquid

Method

Target gene:
Salmonella typhimurium: histidine
Escherichia coli: tryptophan
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 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
Species / strain:
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:
Experiment I: 5000, 1581, 500, 158, 50 and 15.8 μg/plate
Experiment II: 5000; 1581, 500, 158, 50 and 15.8 μg/plate
Confirmatory Experiment II: 5000; 1581, 500, 158, 50 and 15.8 μg/plate
Vehicle:
- 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
Negative controls:
yes
Remarks:
Culture medium
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine (NPD)
Remarks:
Salmonella TA98 (-S9 mix)
Negative controls:
yes
Remarks:
Culture medium
Solvent controls:
yes
Remarks:
Ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Salmonella TA100 and TA1535 (-S9 mix)
Negative controls:
yes
Remarks:
Culture medium
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Salmonella TA1537 (-S9 mix)
Negative controls:
yes
Remarks:
Culture medium
Solvent controls:
yes
Remarks:
Ultrapure water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
E.coli WP2 uvrA (-S9 mix)
Negative controls:
yes
Remarks:
Culture medium
Solvent controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Salmonella TA98, TA100, TA1535, TA 1538 and E.coli WP2 uvrA (+S9 mix)
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min at 37 ºC
- Exposure duration: 48 hours at 37 °C

SELECTION AGENT:
- Salmonella typhimurium: L-Histidine
- Escherichia coli: L-Tryptophan

NUMBER OF REPLICATIONS: Two (Confirmatory Assay and Repeated Confirmatory Assay)

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 results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no, but tested up to limit concentrations
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with strains Salmonella typhimurium TA98 and TA100 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 toxicity test the concentrations examined were: 5000, 1581, 500, 158, 50, 15.8 and 5 μg/plate.
The obtained revertant colony numbers were lower than the revertant colony numbers of the vehicle control (and below the historical control data range) without any biological significance in S. typhimurium TA100 at 5 μg/plate, without metabolic activation (-S9 Mix).
The revertant colony numbers were slightly lower (but remained in the corresponding historical control data range) than the revertant colony numbers of the vehicle control plates in S. typhimurium TA98 at 5 μg/plate, without metabolic activation (-S9 Mix).
Slightly higher revertant colony counts were obtained at 5000 μg/plate in the case of S. typhimurium TA100 (-S9 Mix), in TA98 with and without metabolic activation (±S9 Mix); furthermore at 1581, 50 and 15.8 μg/plate in TA98, with addition of metabolic activation (+S9 Mix).
All of the observed changes remained in the biological variability range of the applied test system.

COMPARISON WITH HISTORICAL CONTROL DATA:
In the Initial Mutation Test the revertant colony numbers were higher than the revertant colony numbers of the vehicle control plates but remained within the corresponding historical control data ranges at 5000 μg/plate in the case of S. typhimurium TA100 and E. coli WP2 uvrA without metabolic activation (-S9 Mix), in TA1535 (±S9 Mix); at 1581 μg/plate in E. coli WP2 uvrA (-S9 Mix); at 500 μg/plate in TA1535 (±S9 Mix); at 158 and 50 μg/plate in TA1535 with addition of metabolic activation (+S9 Mix) and at 15.8 μg/plate in TA1537 (-S9 Mix), in TA1535 (±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 ranges (without any biological significance) in S. typhimurium TA98 at 5000, 158 and 50 μg/plate (-S9 Mix) and at 500 and 15.8 μg/plate (+S9 Mix).
The revertant colony numbers were in the actual vehicle control data range, but were below the historical control data range in S. typhimurium TA100, at 158, 50 and 15.8 μg/plate, without metabolic activation (-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 500 μg/plate in S. typhimurium TA1537 (+S9 Mix) and at the concentrations of 50 and 15.8 μg/plate in E.WP2 uvrA (+S9 Mix).
In the Confirmatory Mutation Test the revertant colony numbers of the positive reference control 2-Aminoanthracene plates (in presence of exogeneous metabolic activation); furthermore the revertant colony numbers of the 9-Aminoacridine plates (in absence of exogeneous metabolic activation) were in the range of the applied dimethyl sulfoxide (DMSO) vehicle control. The following parts of the Confirmatory Mutation Test were considered as invalid: S. typhimurium TA98, TA100, TA1535 and Escherichia coli WP2 uvrA in presence (+S9 Mix), S. typhimurium TA1537 in the presence and also in the absence of exogenous metabolic activation (±S9 Mix). These obtained results were excluded from the evaluation and from the final conclusion of the study.
In the evaluated part of the Confirmatory Mutation Test the revertant colony numbers were higher than the revertant colony numbers of the vehicle control, and were above the corresponding historical control data ranges at 5000 μg/plate in E. coli WP2 uvrA (-S9 Mix), at 1581 μg/plate in S. typhimurium TA98 and in E. coli WP2 uvrA (-S9 Mix).
The obtained increase was unique in the case of S. typhimurium TA98 and was attended by dose relationship in E. coli WP2 uvrA; however the threshold for being positive was not reached in any case.
All of the other observed higher revertant colony numbers compared to vehicle control remained in the historical control data ranges. Higher revertant colony numbers were achieved at 1581 μg/plate in S. typhimurium TA100 and TA1535 (-S9 Mix), at 500 μg/plate in TA98 and E. coli WP2 uvrA (-S9 Mix); furthermore at 158, 50 and 15.8 μg/plate in E. coli WP2 uvrA (-S9 Mix).
In the Confirmatory Mutation Test the lower revertant colony numbers were below the corresponding historical control data range at 5000 μg/plate in S. typhimurium TA98 and TA100 without metabolic activation (-S9 Mix), furthermore at 15.8 μg/plate in TA100, without metabolic activation (-S9 Mix). The low revertant counts at the highest concentration level in S. typhimurium TA98 and TA100 were not evaluated as sign of the unequivocal inhibitory effect of the test item.
In the Repeated Confirmatory Mutation Test the revertant colony numbers were higher than the revertant colony numbers of the vehicle control plates and above the corresponding historical control data range at 5000 μg/plate in S. typhimurium TA98 (+S9 Mix). All of the further revertant colony number increases remained in the historical control data ranges: at 5000 μg/plate in S. typhimurium TA1535 (+S9 Mix); at 1581 μg/plate in TA98, TA100, TA1535 and E. coli WP2 uvrA (+S9 Mix); at 500 μg/plate in TA98 and TA1537 (+S9 Mix); at 158 μg/plate in TA98 (+S9 Mix); at 50 μg/plate in TA98 (+S9 Mix), in TA1537 (-S9 Mix). Most of these increases occurred sporadically; but dose-relationship was noticed in the case of S. typhimurium TA98. The obtained revertant colony number increases remained far below the threshold for being positive in all cases.
Slightly lower revertant colony counts (within the historical control data ranges) were obtained at 158 μg/plate in S. typhimurium TA1537 (-S9 Mix) and at 15.8 μg/plate in TA1535 (+S9 Mix).

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 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 in the Initial Mutation Test, in the Confirmatory Mutation Test in S. typhimurium TA98, TA100, TA1535 and in E. coli WP2 uvrA (-S9 Mix) and in the Repeated Confirmatory Mutation Test.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No biologically relevant increases or substantial decreases (or any unequivocal sign of cytotoxic effect of the test item) were observed in revertant colony numbers of any of the five test strains following treatment with Aldehyde M at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the historical control data ranges were observed in all independently performed main experiments.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The reported data of this 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, Aldehyde M 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 Aldehyde M in independent experiments, in a plate incorporation test (experiment I, Initial Mutation Test), in a pre-incubation test (experiment II, Confirmatory Mutation Test) and in a second pre-incubation test (experiment III, Repeated Confirmatory Mutation Test). The test item was dissolved in dimethyl sulfoxide (DMSO). In the Initial and Confirmatory and Repeated Confirmatory Mutation Tests the tested concentrations were: 5000, 1581, 500, 158, 50 and 15.8 μg/plate.

The Initial and Confirmatory Mutation Tests were conducted with and without metabolic activation (S9 Mix). In the Repeated Confirmatory Mutation Test the Salmonella typhimurium TA98, TA100, TA1535 strains and Escherichia coli WP2 uvrA were investigated in presence of metabolic activation, the Salmonella typhimurium TA1537 in presence and also in absence of metabolic activation. The concentrations, including the controls, were tested in triplicate.

In the performed experiments positive and negative (vehicle) controls were 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 and were within the corresponding historical control data ranges.

The reference mutagens showed a distinct increase of induced revertant colonies in the Initial Mutation Test, in the Repeated Confirmatory Mutation Test and partially in the Confirmatory Mutation Test. In the Confirmatory Mutation Test the revertant colony numbers of the positive reference control 2-Aminoanthracene plates (in presence of exogeneous metabolic activation) and the revertant colony numbers of the 9-Aminoacridine plates (in absence of exogeneous metabolic activation) were in the range of the applied dimethyl sulfoxide (DMSO) vehicle control. The lack of a biologically relevant revertant colony number increase at these positive controls invalidate the experimental parts that had to be repeated (Repeated Confirmatory Mutation Test). These obtained results at the invalid experimental parts were excluded from the evaluation and from the final conclusion of the study.

In the performed experimental phases at least five analyzable concentrations and a minimum of three non-toxic dose levels at each tester strain were applied. The validity criteria of the study were fulfilled.

No biologically relevant increases or substantial decreases (or any unequivocal sign of cytotoxic effect of the test item) were observed in revertant colony numbers of any of the five test strains following treatment with Aldehyde M at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values, mostly within the actual historical control data ranges were observed in both independently performed main experiments.

However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.