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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 05 to November 07, 2016
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected on 05 July 2016 / signed on 28 October 2016
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(5,5-dimethyl-1-cyclohexen-1-yl)pent-4-en-1-one
EC Number:
260-486-7
EC Name:
1-(5,5-dimethyl-1-cyclohexen-1-yl)pent-4-en-1-one
Cas Number:
56973-85-4
Molecular formula:
C13H20O
IUPAC Name:
1-(5,5-dimethylcyclohex-1-en-1-yl)pent-4-en-1-one
Test material form:
liquid
Details on test material:
- Physical state: colourless to pale yellow liquid
- Storage condition of test material: Dry area, ambient temperature (10 to 30 °C) in the dark

Method

Target gene:
Histidine and tryptophan gene for Salmonella typhimurium and Escherichia coli, respectively.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The S9 Microsomal fraction was pre-prepared using standardized in-house procedures (outside the confines of this study). The 10% S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
Test concentrations with justification for top dose:
Experiment 1 (Plate Incorporation Method):
1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix. The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 (Pre-Incubation Method):
- Salmonella strains TA100 and TA1535 (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.
- Salmonella strains TA98 and TA1537 and Escherichia coli strain WP2uvrA (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
Eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.
- Preparation of test materials: The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (8.8%) of the test item. All formulations were used within four hours of preparation and were assumed to be stable for this period. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10^-4 microns.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated: spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Remarks:
untreated: spontaneous mutation rates
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: The bacteria used in the test were obtained from the University of California, Berkeley, and from the British Industrial Biological Research Association.

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes with shaking
- Exposure duration: approximately 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

NUMBER OF REPLICATIONS: Triplicate plates per dose level in experiment 1 and experiment 2.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning.

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Rationale for test conditions:
Experiment 1 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 2 - test item dose levels were selected based on Experiment 1 results in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item.
- Salmonella strains TA100 and TA1535 (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate. The test item was cytotoxic in Salmonella strains TA100 and TA1535 from 500 μg/plate in Experiment 1.
- Salmonella strains TA98 and TA1537 and Escherichia coli strain WP2uvrA (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate. The maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- Fold increases greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration.
- Precipitation: A test item precipitate (globular in appearance) was observed under a low power microscope at 5000 µg/plate.
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: Not applicable


COMPARISON WITH HISTORICAL CONTROL DATA:
The individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.7 times the concurrent vehicle controls.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first mutation test (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns of Salmonella strains TA100 and TA1535 dosed in both the absence and presence of S9-mix from 500 µg/plate and to TA98 and TA1537 at 5000 µg/plate (absence of S9-mix only). No toxicity was noted to Escherichia coli strain WP2uvrA in both the absence or presence of S9-mix and TA98 and TA1537 dosed in the presence of S9-mix. Consequently, the same maximum dose level (5000 µg/plate) or the toxic limit was employed in the second mutation test depending on bacterial strain type. The test item induced a stronger toxic response in the second mutation test, after implementation of the pre-incubation method, with weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate (TA1535), 150 µg/plate (TA100, TA98 and TA1537) and 500 µg/plate (WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were initially noted from 150 µg/plate (TA100), 500 µg/plate (TA1535 and TA1537) and 1500 µg/plate (TA98 and WP2uvrA).

OTHERS:
In both experiments 1 and 2, there were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).
Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 1.5 μg/plate (TA100 -S9-mix) and in the second mutation test at 15 µg/plate (TA1535 -S9-mix) and 500 µg/plate (WP2uvrA +S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility.

- The test material formulation, amino acid supplemented top agar and S9-mix used in this experiment were shown to be sterile.
Remarks on result:
other:
Remarks:
Cytotoxicity Experiment 1: +/-S9 mix: TA100 and TA1535 from 500 µg/plate. +S9 mix: TA98 and TA1537 at 5000 µg/plate. Experiment 2: - S9-mix: TA1535 from 50 µg/plate, TA100, TA98 and TA1537 from 150 µg/plate and WP2uvrA from 500 µg/plate. + S9-mix: TA100 from 150 µg/plate, TA1535 and TA1537 from 500 µg/plate and TA98 and WP2uvrA from 1500 µg/plate.

Any other information on results incl. tables

 Table 7.6.1/2. Mutagenic and cytotoxic effect of the test material.

Strain

S9-mix

Test concentration range

(µg/plate)

Lowest mutagenic concentration (µg/plate)

Lowest cytotoxic

concentration (µg/plate)

 

TA100

-

0.5 - 5000

None

150

+

1.5 - 5000

None

150

TA1535

-

0.5 - 5000

None

50

+

1.5 - 5000

None

500

WP2uvrA

-

1.5 - 5000

None

500

+

1.5 - 5000

None

1500

TA98

-

1.5 - 5000

None

150

+

1.5 - 5000

None

1500

TA1537

-

1.5 - 5000

None

150

+

1.5 - 5000

None

500

Applicant's summary and conclusion

Conclusions:
Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item diluted in DMSO both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the Ames plate incorporation and pre‑incubation methods in Experiment 1 and Experiment 2, respectively.

Experiment 1 (Plate Incorporation Method):

1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix

Experiment 2 (Pre-Incubation Method):

- Salmonella strains TA98 and TA1537 and Escherichia coli strain WP2uvrA (with and without S9-mix): 1.5, 5, 15, 50, 150, 500, 1500, 5000 μg/plate.

- Salmonella strains TA100 and TA1535 (with and without S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.

 

Vehicle (dimethyl sulphoxide) and positive control groups were also included in mutagenicity tests.

 

In the first mutation test (plate incorporation method) the test item induced a visible reduction in the growth of the bacterial background lawns of Salmonella strains TA100 and TA1535 dosed in both the absence and presence of S9-mix from 500 µg/plate and to TA98 and TA1537 at 5000 µg/plate (absence of S9-mix only). No toxicity was noted to Escherichia coli strain WP2uvrA in both the absence or presence of S9-mix and TA98 and TA1537 dosed in the presence of S9-mix. Consequently, the same maximum dose level (5000 µg/plate) or the toxic limit was employed in the second mutation test depending on bacterial strain type. The test item induced a stronger toxic response in the second mutation test, after implementation of the pre-incubation method, with weakened bacterial background lawns noted in the absence of S9-mix from 50 µg/plate (TA1535), 150 µg/plate (TA100, TA98 and TA1537) and 500 µg/plate (WP2uvrA). In the presence of S9-mix, weakened bacterial background lawns were initially noted from 150 µg/plate (TA100), 500 µg/plate (TA1535 and TA1537) and 1500 µg/plate (TA98 and WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.  A test item precipitate (globular in appearance) was observed under a low power microscope at 5000 µg/plate.

 

In both experiments 1 and 2, there were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 1.5 μg/plate (TA100 -S9 -mix) and in the second mutation test at 15 μg/plate (TA1535 -S9 -mix) and 500 µg/plate (WP2uvrA -S9 -mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.7 times the concurrent vehicle controls. Further statistically significant increases in revertant colony frequency were also observed in both the first and second mutation tests, however these increases were considered to have no biological relevance because weakened bacterial background lawns were also noted. Therefore, the responses are considered false and due to additional histidine being available to His- bacteria allowing these cells to undergo several additional cell divisions and presenting as non-revertant colonies.

 

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.

 

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

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