Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
From September 09 to 28, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD test Guideline No. 471 without any deviation
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
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:
UK GLP Compliance Program (inspected on June 27, 2015 / Signed on September 24, 2015)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Juniper, Juniperus mexicana, ext., epoxidized
EC Number:
309-066-8
EC Name:
Juniper, Juniperus mexicana, ext., epoxidized
Cas Number:
99811-75-3
Molecular formula:
not applicable (UVCB substance)
IUPAC Name:
(1S,2R,5S,7R)-2,6,6,8-tetramethyltricyclo[5.3.1.0¹,⁵]undecan-9-one; (1aS,2R,4aS,8aS)-2,4a,8,8-tetramethyl-decahydrocyclopropa[e]naphthalen-3-one; (1aS,2S,4aS,8aS)-2,4a,8,8-tetramethyl-decahydrocyclopropa[e]naphthalen-3-one
Test material form:
other: liquid
Details on test material:
- Physical state: Pale yellow liquid
- Storage condition of the test material: Room temperature, 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
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10% S9-mix : S9 from the livers of male rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day by oral route, for 3 days prior to preparation on day 4).
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
Experiment 2 (Plate Incorporation Method):
50, 150, 250, 500, 1000, 1500, 3000, 5000 μg/plate in all strains with and without S9-mix.
A number of intermediate concentration levels were included to try and attain a clear, dose-related response following the observation of statistically significant increases in revertant colony frequency in the first mutation test and also to achieve a minimum of four non-toxic doses for the relevant strains that showed toxicity in the first mutation test.
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. 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
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
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)
DURATION
- Exposure duration: Plates were incubated at 37 °C ± 3 °C for approximately 48 hours
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. Several manual counts were required due to bubbles in the base agar and colonies on the edge of the plates, slightly distorting the actual plate count.
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 analysis of data as determined by UKEMS (Mahon et al., 1989).

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:
positive
Remarks:
Weakly positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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 was immiscible in the sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration.
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA:
In both experiments 1 and 2, small but toxicologically significant increases in the revertant colony frequency of TA100, TA1535 (absence and presence of S9-mix) and WP2uvrA (absence of S9-mix only) at the upper test item dose levels.
In Experiment 2, a clear dose-related response in WP2uvrA frequency between 1000 and 5000 μg/plate in the absence of S9-mix was noted. The increases over the concurrent vehicle control for this strain were 2.5 times at 1500 μg/plate, 4.1 times at 3000 μg/plate and 4.7 times at 5000 μg/plate (mean numbers). Furthermore, the individual revertant colony counts at 3000 and 5000 μg/plate exceeded the in-house historical control range for the bacterial tester strain. Any excursions outside the maxima ranges, particularly when reproducibility is apparent (5000 μg/plate in Experiments 1 and 2), must be considered to be evidence of a biological response. There was no evidence of a response with WP2uvrA dosed in the presence of S9-mix, this lack of activity may be due to partial inhibition following metabolic activation.
Smaller responses were also noted to TA100 (1.5 times at 5000 μg/plate in the absence of S9-mix and 1.4 times at 5000 μg/plate in the presence of S9-mix) ) and TA1535 (1.6 times at 1000 μg/plate and 3.0 times at 1500 μg/plate in the absence of S9-mix. 1.8 times at 500 and 1500 μg/plate, 2.0 at 1000 μg/plate - in the presence of S9-mix), although there was a slight shift in toxicity to TA1535 in the second mutation test with weakened lawns initially noted at 1500 μg/plate compared to 5000 μg/plate in the first mutation test.
A small increase in TA98 revertant colony frequency was also noted at and above 3000 μg/plate (absence of S9-mix) in the second mutation test, however this increase was non-reproducible over the two experiments.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies generally
within the normal range. A small number of vehicle control revertant colony counts for TA100 and TA1535 (dosed in the absence or presence of S9-mix after the first mutation test) and TA1537 (dosed in the presence of S9-mix in the second mutation test) were either just below or above the historical control ranges. These counts were still considered acceptable as the other vehicle and untreated control counts were within expected range and each tester strain responded very well with the respective positive controls in both the presence and absence of S9-mix. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The test item was cytotoxic in Salmonella strains TA1535 and TA1537 at 5000 μg/plate and from 1500 μg/plate in Experiments 1 and 2, respectively, in both the presence and absence of metabolic activation (S9-mix). No toxicity was noted to Salmonella strains TA100 and TA98 and Escherichia coli strain WP2uvrA.

OTHERS:
- 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: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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

-

1.5 - 5000

5000

None

+

1.5 - 5000

5000

None

TA1535

-

1.5 - 5000

1000

1500

+

1.5 - 5000

500

1500

WP2uvrA

-

1.5 - 5000

1000

None

+

1.5 - 5000

None

None

TA98

-

1.5 - 5000

3000

None

+

1.5 - 5000

None

None

TA1537

-

1.5 - 5000

None

1500

+

1.5 - 5000

None

1500

See the attached document for more information on tables of results.

Applicant's summary and conclusion

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

Under the test conditions, test material is mutagenic with and without metabolic activation in S. typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).
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 plate incorporation method in two independent experiments. The dose range for Experiment 1 was 1.5 to 5000 mg/plate in all the tester strains, with and without S9-mix.  The dose range for Experiment 2 was 50 to 5000 µg/plate in all the tester strains, with and without S9-mix, based on the results of expriment 1.

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

 

The test item was cytotoxic in Salmonella strains TA1535 and TA1537 at 5000 μg/plate and from 1500 μg/plate in Experiments 1 and 2, respectively, in both the presence and absence of metabolic activation (S9-mix). No toxicity was noted to Salmonella strains TA100 and TA98 and Escherichia coli strain WP2uvrA. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

 

In both experiments 1 and 2, small but toxicologically significant increases in the revertant colony frequency of TA100, TA1535 (absence and presence of S9-mix) and WP2uvrA (absence of S9-mix only) at the upper test item dose levels.

In Experiment 2, a clear dose-related response in WP2uvrA frequency between 1000 and 5000 μg/plate in the absence of S9-mix was noted. The increases over the concurrent vehicle control for this strain were 2.5 times at 1500 μg/plate, 4.1 times at 3000 μg/plate and 4.7 times at 5000 μg/plate (mean numbers). Furthermore, the individual revertant colony counts at 3000 and 5000 μg/plate exceeded the in-house historical control range for the bacterial tester strain. Any excursions outside the maxima ranges, particularly when reproducibility is apparent (5000 μg/plate in Experiments 1 and 2), must be considered to be evidence of a biological response. There was no evidence of a response with WP2uvrA dosed in the presence of S9-mix, this lack of activity may be due to partial inhibition following metabolic activation.

Smaller responses were also noted to TA100 (1.5 times at 5000 μg/plate in the absence of S9-mix and 1.4 times at 5000 μg/plate in the presence of S9-mix) ) and TA1535 (1.6 times at 1000 μg/plate and 3.0 times at 1500 μg/plate in the absence of S9-mix. 1.8 times at 500 and 1500 μg/plate, 2.0 at 1000 μg/plate - in the presence of S9-mix), although there was a slight shift in toxicity to TA1535 in the second mutation test with weakened lawns initially noted at 1500 μg/plate compared to 5000 μg/plate in the first mutation test.

A small increase in TA98 revertant colony frequency was also noted at and above 3000 μg/plate (absence of S9-mix) in the second mutation test, however this increase was non-reproducible over the two experiments.

 

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies generally within the normal range. A small number of vehicle control revertant colony counts for TA100 and TA1535 (dosed in the absence or presence of S9-mix after the first mutation test) and TA1537 (dosed in the presence of S9-mix in the second mutation test) were either just below or above the historical control ranges. These counts were still considered acceptable as the other vehicle and untreated control counts were within expected range and each tester strain responded very well with the respective positive controls in both the presence and absence of S9-mix. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

 

Under the test conditions, test material is mutagenic with and without metabolic activation in S. typhimurium (strains TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

 

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