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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:
October 22 to November 17, 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
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:
2015
Report date:
2015

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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Resinoid of Abies balsamea (Pinaceae) obtained from needles by an organic solvent extraction
EC Number:
947-304-8
Molecular formula:
not applicable for UVCB
IUPAC Name:
Resinoid of Abies balsamea (Pinaceae) obtained from needles by an organic solvent extraction
Test material form:
other: resin
Details on test material:
- Physical state: black/dark green resin
- Storage condition of test material: Room temperature in the dark

Method

Target gene:
Histidine and tryptophan.
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: S9-mix from the livers of male rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day by oral route).
Test concentrations with justification for top dose:
Test for Mutagenicity (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
Test for Mutagenicity (Experiment 2) – Plate Incorporation Method: 150, 500, 1500, 2000, 3000, 4000 and 5000 μg/plate in all strains with and without S9-mix
Test for Mutagenicity (Experiment 3) – Plate Incorporation Method: 50, 150, 500, 1500 and 5000 µg/plate in TA 100 strain with and without S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL and acetone at 100 mg/mL but was fully soluble in tetrahydrofuran at 200 mg/mL in solubility checks performed in-house. Tetrahydrofuran was therefore selected as the vehicle.
- Preparation of test materials: The test item was accurately weighed and appropriate dilutions prepared in tetrahydrofuran by mixing on a vortex mixer and sonication for 20 minutes at 40 °C on the day of each experiment. Tetrahydrofuran is toxic to the bacterial cells at and above 50 μL (0.05 mL), therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 μL (0.025 mL) aliquots. Tetrahydrofuran is considered an acceptable vehicle for use in this test system (Maron et al., 1981). All formulations were used within four hours of preparation and were assumed to be stable for this period.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
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:
Tetrahydrofuran
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:
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.

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

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system. Manual counts were performed at 5000 μg/plate because of test item precipitation. A further manual count was also required, due to interference in the base agar e.g. marks on the base of the plates slightly distorting the 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.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

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:
no cytotoxicity, but tested up to precipitating concentrations
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: None
- Precipitation: A test item precipitate (globular, greasy and black particulate in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2013 and 2014 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the second mutation test and the confirmatory test (TA100 only).

OTHERS:
- The test material formulation, amino acid supplemented top agar and S9-mix used in this experiment were shown to be sterile.

Any other information on results incl. tables

See the attached document for information on tables of results

Applicant's summary and conclusion

Conclusions:
negative with metabolic activation
negative without metabolic activation

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- according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and of the Directive 67/548/EEC.
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 TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test material both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).

Test for Mutagenicity (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

Test for Mutagenicity (Experiment 2) – Plate Incorporation Method: 150, 500, 1500, 2000, 3000, 4000 and 5000 μg/plate in all strains with and without S9-mix

Test for Mutagenicity (Experiment 3) – Plate Incorporation Method: 50, 150, 500, 1500 and 5000 µg/plate in TA 100 strain with and without S9-mix

Negative, vehicle (tetrahydrofuran) and positive control groups were also included in mutagenicity tests.

The vehicle 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.

 

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation in any of the experiment. A test item precipitate (globular, greasy and black particulate in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

 

In the first mutation test, small but consistent increases in TA100 revertant colony frequency were noted, particularly in the absence of S9-mix, at 5000 μg/plate. The increases achieved a maximum 2.1 fold increase over the concurrent vehicle control and individual revertant counts were above the in-house maxima for the tester strain. However, no significant increases in the frequency of TA100 revertant colonies were noted at any of the test item dose levels either with or without S9-mix in Experiment 2 and Experiment 3 (confirmatory test). The test item was considered to be non-mutagenic to Salmonella strain TA100 because there were no reproducible increases in revertant colony frequency noted in three separate experiments even after the inclusion of intermediate dose levels in Experiment 2. No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the remaining bacterial strains, with any dose of the test item, either with or without metabolic activation. A small, statistically significant increase in TA1537 revertant colony frequency was observed in the absence of S9-mix at 150 μg/plate in the second mutation test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 150 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.8 times the concurrent vehicle control.

 

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- according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) .

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