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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:
16 January - 25 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 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:
2013
Report date:
2013

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 Programme (inspected on 10 July 2012/ signed on 30 November 2012)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Juniper, Juniperus communis, ext.
EC Number:
283-268-3
EC Name:
Juniper, Juniperus communis, ext.
Cas Number:
84603-69-0
Molecular formula:
Not applicable for UVCB
IUPAC Name:
Juniper, Juniperus communis, ext.
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Juniper oil
- Physical state: Extremely pale yellow liquid
- Analytical purity: 100 %
- Date received: 28 November 2012
- Expiration date of the lot/batch: 28 November 2014
- Storage condition of test material: Room temperature in the dark

Method

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 fraction prepared from liver homogenates of rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg bw/day by oral route
Test concentrations with justification for top dose:
Preliminary Toxicity Test (preincubation method):
- 50, 150, 500, 1500 and 5000 μg/plate in TA100 or WP2uvrA strains, presence and absence of S9- mix.

Mutation Test:
Experiment 1 (preincubation method):
- Salmonella strains TA 98 and TA 1537 (absence and presence of S9-mix), TA 100 (presence of S9-mix) and TA 1535 (absence of S9-mix): 0.0005, 0.0015, 0.005, 0.015, 0.05, 0.15 and 0.5 μg/plate.
- Salmonella strain TA 100 (absence of S9-mix): 0.0015, 0.005, 0.015, 0.05, 0.15, 0.5 and 1.5 μg/plate.
- Salmonella strain TA 1535 (presence of S9-mix): 0.005, 0.015, 0.05, 0.15, 0.5, 1.5 and 5 μg/plate.
- E.coli strain WP2uvrA (absence and presence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate.

Experiment 2 (preincubation method):
- Salmonella strains TA 98, TA 1535 and TA 1537 (absence of S9-mix) and TA 100 (presence and absence of S9-mix): 0.0015, 0.005, 0.015, 0.05, 0.15, 0.5 and 1.5 μg/plate.
- Salmonella strains TA 98 and TA 1537 (presence of S9-mix): 0.0005, 0.0015, 0.005, 0.015, 0.05, 0.15 and 0.5 μg/plate.
- Salmonella strain TA 1535 (presence of S9-mix): 0.005, 0.015, 0.05, 0.15, 0.5, 1.5 and 5 μg/plate.
- E.coli strain WP2uvrA (absence and presence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: Test item was immiscible in sterile distilled water, dimethyl sulphoxide, dimethyl formamide at 50 mg/mL and acetone at 100 mg/mL but was fully miscible in tetrahydrofuran at 200 mg/mL in solubility checks performed in-house. Tetrahydrofuran was therefore selected as the vehicle.
- Formulation preparation: Test item was accurately weighed and approximate half-log dilutions prepared in tetrahydrofuran 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.
Controlsopen allclose all
Untreated negative controls:
no
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 metabolic activation
Untreated negative controls:
no
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 metabolic activation
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: All strains of bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 or from the British Industrial Biological Research Association, on nutrient agar plates, on 17 August 1987.

METHOD OF APPLICATION: Preincubation (test item, vehicle and positive controls) and plate incorporation (negative control) methods

DURATION
- Preincubation period: 20 minutes at 37 °C with shaking at approximately 130 rpm
- Incubation period: Treated plates were placed in anaerobic jars or bags (one jar/bag for each concentration of test item/vehicle) and incubated at 37 °C for approximately 48 h.

NUMBER OF REPLICATIONS:
-1 plate/dose for preliminary toxicity test and 3 plates/dose for treatment, negative, vehicle and positive controls in mutation test (Experiment 1 & 2).

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity was determined on the basis of growth of the bacterial background lawn.

OTHER: After approximately 48 h incubation at 37 °C the plates were assessed for numbers of revertant colonies using an automated colony counter.
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 increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).
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 judgement 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
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
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- 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.

PRELIMINARY TOXICITY TEST:
- The test item initially exhibited toxicity to TA 100 from 50 µg/plate and 1500 µg/plate to WP2uvrA.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Mutation Test: The test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains, initially from 0.15 µg/plate in both the absence and presence of S9-mix. The test item also induced toxicity to Escherichia coli strain WP2uvrA, initially from 15 µg/plate in the absence of S9-mix and 50 µg/plate in the presence of S9-mix. The sensitivity of the tester strains to the toxicity of the test item varied both between strain type, exposures with or without S9-mix and experiment number.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Results were compared with historical negative, solvent and positive control data (2010 and 2011) and lies within the range.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile.
- The culture density for each bacterial strain was also checked and considered acceptable.
- Test item formulation used in this experiment was shown to be sterile.

Any other information on results incl. tables

Table 7.6.1/3: Preliminary Toxicity Test : number of revertants depending on the tested concentration

With (+) or

without (-)

S9-mix

Strain

 

Dose (µg/plate)

0

50

150

500

1500

5000

-

TA 100

124

82 S

76 V

83 V

0 V

0 V

+

TA 100

95

86 S

89 S

73 S

0 V

0 V

-

WP2 uvr A

30

26

24

30

30 S

28 S

+

WP2 uvr A

29

24

25

27

29 S

41 S

 

S: sparse bacterial background lawn

V: Very weak bacterial background lawn

See the attached Document for information on tables of results - Main experiments

Applicant's summary and conclusion

Conclusions:
Under the test condition, Juniper oil is not mutagenic with and without metabolic activation to strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli WP2 uvr A.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and Escherichia coli (WP2uvrA) were exposed to Juniper oil at the following concentrations under anaerobic conditions:

 

Preliminary Toxicity Test (preincubation method):

- 50, 150, 500, 1500 and 5000 μg/plate in TA100 or WP2uvrA strains, presence and absence of S9- mix.

 

Mutation Test:

Experiment 1 (preincubation method):

-Salmonella strains TA 98 and TA 1537 (absence and presence of S9-mix), TA 100 (presence of S9-mix) and TA 1535 (absence of S9-mix): 0.0005, 0.0015, 0.005, 0.015, 0.05, 0.15 and 0.5 μg/plate.

-Salmonella strain TA 100 (absence of S9-mix): 0.0015, 0.005, 0.015, 0.05, 0.15, 0.5 and 1.5 μg/plate.

-Salmonella strain TA 1535 (presence of S9-mix): 0.005, 0.015, 0.05, 0.15, 0.5, 1.5 and 5 μg/plate.

-E.coli strain WP2uvrA (absence and presence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate.

 

Experiment 2 (preincubation method):

-Salmonella strains TA 98, TA 1535 and TA 1537 (absence of S9-mix) and TA 100 (presence and absence of S9-mix): 0.0015, 0.005, 0.015, 0.05, 0.15, 0.5 and 1.5 μg/plate.

-Salmonella strains TA 98 and TA 1537 (presence of S9-mix): 0.0005, 0.0015, 0.005, 0.015, 0.05, 0.15 and 0.5 μg/plate.

-Salmonella strain TA 1535 (presence of S9-mix): 0.005, 0.015, 0.05, 0.15, 0.5, 1.5 and 5 μg/plate.

-E.coli strain WP2uvrA (absence and presence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate.

 

Metabolic activation system used in this test was10 % S9; S9 fraction prepared from liver homogenates of rats induced with Phenobarbitone/β-Naphthoflavone. Negative, vehicle and positive control groups were also included in mutagenicity tests.

 

In preliminary toxicity test, the test item initially exhibited toxicity to TA 100 from 50 µg/plate and 1500 µg/plate to WP2uvrA. In main experiments, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella tester strains, initially from 0.15 µg/plate in both the absence and presence of S9-mix. The test item also induced toxicity to Escherichia coli strain WP2uvrA, initially from 15 µg/plate in the absence of S9-mix and 50 µg/plate in the presence of S9-mix. The sensitivity of the tester strains to the toxicity of the test item varied both between strain type, exposures with or without S9-mix and experiment number. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No biologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation. Small, statistically significant increases in TA 98 revertant colony frequency were observed in Experiment 2 at 0.0005, 0.0015, 0.015 and 0.5 µg/plate in the absence of S9-mix only. 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 the tester strain and the maximum fold increase was only 1.6 times the concurrent vehicle control.The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study. 

 

Under the test conditions, Juniper oil is not considered as mutagenic in these bacterial systems.

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

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