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:
16 January - 26 March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD Guideline 471 without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
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. certificate)
Remarks:
UK GLP Compliance Programme (inspected on 10 July 2012/ signed on 30 November 2012)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Pine needle oil
- Physical state: Colourless 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

Target gene:
Histidine and tryptophan gene
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. As toxicity was observed from 50 µg/plate a second preliminary test was performed.
- 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate in TA100 or WP2uvrA strains, presence and absence of S9- mix.

Mutation Test:
Experiment 1 (preincubation method):
- All Salmonella strains (absence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate.
- WP2uvrA (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate.
- All tester strains (presence of S9-rnix): 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.

Experiment 2 (preincubation method):
- All tester strains (presence of S9 mix): 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
- All tester strains (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: Test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in-house. Acetone was therefore selected as the vehicle.
- Formulation preparation: Test item was accurately weighed and approximate half-log dilutions prepared in acetone 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:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
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:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: A light, globular test item precipitate was observed at 5000 µg/plate. Due to the toxicity of the test item this observation was only noted in the preliminary toxicity test.

PRELIMINARY TOXICITY TEST:
- The test item initially exhibited toxicity to the strains of bacteria (TA 100 and WP2uvrA) from 15 and 150 µg/plate in the absence and presence of S9-mix respectively.

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 tester strains, initially from 15 µg/plate in the absence of S9-mix and 150 µg/plate in the presence of S9-mix. The sensitivity of the tester strains to the toxicity of the test item varied slightly between strain type and exposures with or without S9-mix.

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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

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

With (+) or

without (-)

S9-mix

Strain

Dose (µg/plate)

0

0.015

0.05

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA 100

94

80

101

83

90

93

75

76

50V

N/T

N/T

N/T

N/T

+

TA 100

90

N/T

N/T

N/T

N/T

N/T

N/T

N/T

87

57 S

63 S

49 S

59 VP

-

WP2 uvr A

17

19

17

14

11

16

16

15 S

8 S

N/T

N/T

N/T

N/T

+

WP2 uvr A

26

N/T

N/T

N/T

N/T

N/T

N/T

N/T

20

17 S

27 S

12 S

13 SP

S: Sparse bacterial background lawn

V: Very weak bacterial background lawn

N/T: Assay repeated and not tested at this dose level due to toxicity

P: Precipitate

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

Applicant's summary and conclusion

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

Under the test condition, Pine needle 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 Pine needle oil at the following concentrations:

 

Preliminary Toxicity Test (preincubation method): 
- 50, 150, 500, 1500 and 5000 μg/plate in TA100 or WP2uvrA strains, presence and absence of S9- mix.
As toxicity was observed from 50 µg/plate a second preliminary test was performed.
- 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate in TA100 or WP2uvrA strains, presence and absence of S9- mix.

Mutation Test:
Experiment 1 (preincubation method): 
- All Salmonella strains (absence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate.
- WP2uvrA (absence of S9-mix): 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 µg/plate.
- All tester strains (presence of S9-rnix): 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.

Experiment 2 (preincubation method): 
- All tester strains (presence of S9 mix): 0.5, 1.5, 5, 15, 50, 150 and 500 µg/plate.
- All tester strains (absence 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 the strains of bacteria (TA 100 and WP2uvrA) from 15 and 150 µg/plate in the absence and presence of S9-mix respectively. In main experiments, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially from 15 µg/plate in the absence of S9-mix and 150 µg/plate in the presence of S9-mix. The sensitivity of the tester strains to the toxicity of the test item varied slightly between strain type and exposures with or without S9-mix. A light, globular test item precipitate was observed at 5000 µg/plate. Due to the toxicity of the test item this observation was only noted in the preliminary toxicity test. No toxicologically 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. A small, statistically significant increase in TA 98 revertant colony frequency was observed in the absence of S9-mix at 1.5 µg/plate in Experiment 1. 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 1.5 µg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.70 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, Pine needle oil is not considered as mutagenic in these bacterial systems.