Juniper, Juniperus oxycedrus, ext.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 05 to March 13, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

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:

March 05, 2015

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Juniper, Juniperus oxycedrus, ext.

EC Number:

289-969-0

EC Name:

Juniper, Juniperus oxycedrus, ext.

Cas Number:

90046-02-9

IUPAC Name:

Empyreumatic oil obtained from Juniperus oxycedrus (Cupressaceae) wood by dry distillation

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Cade Oil
- Physical state: On the certificate of analysis: dark brown oily liquid; on the test item information sheet: dark brown liquid; at receipt: brown liquid
- Date of receipt: 19 December 2014
- Expiration date of the lot/batch: November 2019
- Storage condition of test material: At room temperature (15-20 °C ± 2 °C) protected from light

Method

Target gene:

Histidine

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

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 liver of rats treated with Aroclor 1254 (500 mg/kg bw) by the intraperitoneal route

Test concentrations with justification for top dose:

Preliminary toxicity test: 10, 100, 500, 1000, 2500 and 5000 μg/plate in the TA 98, TA 100 and TA 102 strains with and without S9-mix, using plate incorporation method.

First experiment (plate incorporation method):
312.5, 625, 1250, 2500 and 5000 μg/plate in the TA 98 and TA 102 strains, without S9-mix
78.1, 156.3, 312.5, 625, 1250 and 2500 μg/plate in the TA 100 strain, without S9-mix
1.3, 2.6, 7.7, 23.1, 69.4, 208.3 and 625 μg/plate in the TA 1535 and TA 1537 strains, without S9-mix
312.5, 625, 1250, 2500 and 5000 μg/plate in the TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains, with S9-mix

Second experiment:
without S9 mix (plate incorporation method):
156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate in the TA 98 and TA 102 strains
78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate in the TA 100 strain
7.8, 15.6, 31.3, 62.5, 125, 250 and 500 μg/plate in the TA 1535 and TA 1537 strains

With S9 mix (pre-incubation method):
39.1, 78.1, 156.3, 312.5, 625 and 1250 μg/plate in the TA 1535 and TA 1537 strains
156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate in the TA 98, TA 100 and TA 102 strains

Third experiment (pre-incubation method):
156.3, 234.4, 312.5, 468.8, 625, 1250 and 2500 μg/plate in the TA 98 strain, with S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Dose formulation preparation
The test item was dissolved in the vehicle at concentrations of:
100 mg/mL for the preliminary toxicity test; 100 mg/mL for the first experiment; 50 and 100 mg/mL for the second experiment; 50 mg/mL for the third experiment.
The stock solutions and their dilutions were prepared within 4 hours of use, and then kept at room temperature and protected from light until use

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Anthramine

Remarks:

with metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: Five strains of Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were supplied by Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) or Culture Collections (Public Health England, Porton Down, Salisbury SP4 0JG, UK), depending on the strain.

METHOD OF APPLICATION: In agar (direct plate incorporation and preincubation method)

DURATION
- Preincubation period: 60 minutes at 37 °C
- Incubation period: Plates were incubated at 37 °C for 48-72 hours

NUMBER OF REPLICATIONS:
- SIngle plate/dose for preliminary toxicity test
- 3 plates/dose for main experiments

DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of the toxicity was performed on the basis of the observation of a decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

OTHER:
- Number of revertants per plate was scored for each strain and for each experimental point using an automatic counter (Sorcerer Automatic Colony Counter for the scoring of colonies and Ames Study Manager for the data management, Perceptive Instruments Ltd, Bury St Edmunds IP33 3TA, UK). Also, the thinning of the bacterial lawn and the presence of precipitate were evaluated

Evaluation criteria:

In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.
The test item is considered to have shown mutagenic activity in this study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose-level,
- a reproducible dose-response relationship is evidenced.
The test item is considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose-levels,
- nor any evidence of a dose-response relationship is noted

Statistics:

None

Results and discussion

Test results

Key result

Species / strain:

other: TA 1535, TA 100, TA 102, TA 1537 and TA 98

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
- Precipitation: None

PRELIMINARY TOXICITY TEST
- A moderate emulsion was observed in the Petri plates when scoring the revertants at 5000 μg/plate.
- A moderate toxicity was noted at 5000 μg/plate towards the TA 98 and TA 100 strains in the absence of S9 mix (decrease in the number of revertants and/or thinning of the bacterial lawn) and in the TA 98 strain in the presence of S9 mix (decrease in the number of revertants). No noteworthy toxicity was noted in the TA 102 strain.

COMPARISON WITH HISTORICAL CONTROL DATA: Results were compared with historical data (September 01, 2013 to March 01, 2014)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiments without S9 mix:
- A moderate emulsion was observed in the Petri plates when scoring the revertants at 5000 μg/plate.
- A moderate to strong toxicity was noted at dose-levels ≥ 208.3 μg/plate in the TA 1535 and TA 1537 strains, ≥ 1250 μg/plate in the TA 98 and 100 strains and at 5000 μg/plate in the TA 102 strain.
Experiments with S9 mix:
- A moderate emulsion was observed in the Petri plates when scoring the revertants at 5000 μg/plate.
- A moderate to strong toxicity was noted at dose-levels ≥ 2500 μg/plate in the TA 1535 and TA 1537 strains and at 5000 μg/plate in the TA 100 strain, in the first experiment (direct plate incorporation method).
- A moderate to strong toxicity was noted at dose-levels ≥ 625 μg/plate in the TA 1535 and TA 1537 strains, ≥ 1250 μg/plate in the TA 98 strain, ≥ 2500 μg/plate in the TA 100 strain and at 5000 μg/plate in the TA 102 strain, in the second and third experiments (pre-incubation method).

MUTAGENICITY:
Experiments without S9 mix
- The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment.
Experiments with S9 mix
- A slight increase in the number of revertant colonies was noted at 312.5 μg/plate in the TA 98 strain in the second experiment (pre-incubation method). This increase reached the positive threshold of 2-fold the vehicle control value. However, the mean number of revertants remained within the historical data range of the corresponding vehicle control (45.0 versus [18-53]). Moreover, there was no clear evidence of a dose-response relationship. Since no noteworthy increase in the number of revertants was observed either in the first experiment (direct plate incorporation method) or in the third experiment using the same experimental conditions (pre-incubation method), despite the use of a narrower range of dose-levels, the slight increase noted in the second experiment was considered to be not biologically relevant.
The test item did not induce any other noteworthy increase in the number of revertants, in any other strains, in either experiment with S9 mix.

Any other information on results incl. tables

None

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, TA100 and TA 102).

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, TA 100 and TA 102 were exposed to test material both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).

Preliminary toxicity test: 10, 100, 500, 1000, 2500 and 5000 μg/plate in the TA 98, TA 100 and TA 102 strains with and without S9-mix, using plate incorporation method

 

First experiment (plate incorporation method):

312.5, 625, 1250, 2500 and 5000 μg/plate in the TA 98 and TA 102 strains, without S9-mix

78.1, 156.3, 312.5, 625, 1250 and 2500 μg/plate in the TA 100 strain, without S9-mix

1.3, 2.6, 7.7, 23.1, 69.4, 208.3 and 625 μg/plate in the TA 1535 and TA 1537 strains, without S9-mix

312.5, 625, 1250, 2500 and 5000 μg/plate in the TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains, with S9-mix

 

Second experiment:

without S9 mix (plate incorporation method):

156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate in the TA 98 and TA 102 strains

78.1, 156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate in the TA 100 strain

7.8, 15.6, 31.3, 62.5, 125, 250 and 500 μg/plate in the TA 1535 and TA 1537 strains 

With S9 mix (pre-incubation method):

39.1, 78.1, 156.3, 312.5, 625 and 1250 μg/plate in the TA 1535 and TA 1537 strains

156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate in the TA 98, TA 100 and TA 102 strains

 

Third experiment (pre-incubation method):

156.3, 234.4, 312.5, 468.8, 625, 1250 and 2500 μg/plate in the TA 98 strain, with S9-mix

 

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

 

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

 

Experiments without S9 mix: A moderate emulsion was observed in the Petri plates when scoring the revertants at 5000 μg/plate. A moderate to strong toxicity was noted at dose-levels≥208.3 μg/plate in the TA 1535 and TA 1537 strains,≥1250 μg/plate in the TA 98 and 100 strains and at 5000 μg/plate in the TA 102 strain. The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment.

 

Experiments with S9 mix: A moderate emulsion was observed in the Petri plates when scoring the revertants at 5000 μg/plate. A moderate to strong toxicity was noted at dose-levels≥2500 μg/plate in the TA 1535 and TA 1537 strains and at 5000 μg/plate in the TA 100 strain, in the first experiment (direct plate incorporation method). A moderate to strong toxicity was noted at dose-levels≥625 μg/plate in the TA 1535 and TA 1537 strains,≥1250 μg/plate in the TA 98 strain,≥2500 μg/plate in the TA 100 strain and at 5000 μg/plate in the TA 102 strain, in the second and third experiments (pre-incubation method).

A slight increase in the number of revertant colonies was noted at 312.5 μg/plate in the TA 98 strain in the second experiment (pre-incubation method). This increase reached the positive threshold of 2-fold the vehicle control value. However, the mean number of revertants remained within the historical data range of the corresponding vehicle control (45.0versus[18-53]). Moreover, there was no clear evidence of a dose-response relationship. Since no noteworthy increase in the number of revertants was observed either in the first experiment (direct plate incorporation method) or in the third experiment using the same experimental conditions (pre-incubation method), despite the use of a narrower range of dose-levels, the slight increase noted in the second experiment was considered to be not biologically relevant. The test item did not induce any other noteworthy increase in the number of revertants, in any other strains, in either experiment with S9 mix.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98, TA100 and TA 102).