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:
5 April - 1 May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to 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:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 4-isopropylidene-1-methylcyclohexene and 1-isopropyl-4-methyl-7-oxabicyclo[2.2.1]heptane and 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane
EC Number:
938-945-4
Molecular formula:
Not applicable
IUPAC Name:
Reaction mass of 4-isopropylidene-1-methylcyclohexene and 1-isopropyl-4-methyl-7-oxabicyclo[2.2.1]heptane and 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octane
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Name of test material (as cited in study report): Terpinolene multiconstituent
- Physical state: Yellow liquid
- Analytical purity: 66.9 % (sum of the three main constituents)
- Composition of test material (%): Terpineols (4.4 %), alpha pinene (1.1 %), alpha fenchene (0.2 %), camphene (1.0 %), alpha phellandrene (0.5 %), alpha terpinene (2.7 %), cineol 1.4 (20.5 %), d-limonene (9.1 %), l-limonene (9.1 %), beta phellandrene (0.2 %), paracymene (0.7 %), cineol 1.8 (14.6 %), gamma terpinene (3.6%), terpinolene (31.8 %) and others (0.5 %)
- Lot/batch No.: 123238
- Date of receipt: 21 February 2012
- Expiration date of the lot/batch: 29 September 2012
- Storage condition of test material: Stored at 2-8 °C, under nitrogen and protected from light

Method

Target gene:
Histidine gene
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:
other: histidine dependent, rfa and uvrB character, resistant to ampicillin or ampicillin plus tetracycline
Metabolic activation:
with and without
Metabolic activation system:
10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254 from Molecular Toxicology Incorporated, USA
Test concentrations with justification for top dose:
Mutagenicity tests:
- Experiment 1: 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate, with and without S9 mix in all 5 strains (plate incorporation method)
- Experiment 2: 4.096, 10.24, 25.6, 64, 160 and 400 μg/plate, with S9 mix (preincubation method) and without S9 mix (plate incorporation method) in all 5 strains [1.638 μg/plate employed for treatment of all strains in the absence of S9 and strains TA 100 and TA 1537 in the presence of S9 only; 1000 μg/plate employed for treatment of strains TA 98, TA 1535 and TA 102 in the presence of S9 only.]
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: Terpinolene multiconstituent was soluble in ethanol at concentrations of at least 502 mg/mL
Formulation procedure:
- Test article stock solutions were prepared by formulating Terpinolene multiconstituent in ethanol under subdued lighting conditions with the aid of vortex mixing where required, immediately prior to assay to give the maximum required treatment solution concentration. Subsequent dilutions were made using ethanol. The test article solutions were protected from light and used within approximately 6 h of initial formulation.

Volume addition: 0.1 mL volume additions of vehicle/test article solution were used for all plate-incorporation treatments, 0.05 mL volume additions of vehicle/test article solution were used for pre-incubation treatments.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Remarks:
See table 7.6.1/1
Positive control substance:
benzo(a)pyrene
other: 2- Aminoanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: Strains TA 98, TA 1535 and TA 1537 were originally obtained from the UK NCTC. Strains TA 100 and TA 102 were derived from cultures originally obtained from Covance Laboratories Inc., USA.

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

DURATION
- Preincubation period: 20 minutes at 37 ± 1 °C
- Incubation period: Plates were inverted and incubated at 37 ± 1 °C in the dark for 3 days.

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS:
- Treatment and positive control groups: 3 plates/dose
- Vehicle control group: 5 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Background bacterial lawn was inspected for signs of toxicity.

OTHER:
Colony counting: Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as bubbles or splits in the agar affected the accuracy of the automated counter.
Evaluation criteria:
- For valid data, the test article was considered to be mutagenic if:
1. When assessed using Dunnett's test, an increase in revertant numbers gave a significant response (p ≤ 0.01) which was concentration related.
2. The positive trend/effects described above were reproducible.

Test article was considered negative in this assay if none of the above criteria were met.
Statistics:
- Dunnett's test was used to compare the counts at each concentration with the control. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 μg/plate).

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
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:
COMPARISON WITH HISTORICAL CONTROL DATA: Mean vehicle control counts fell within the normal historical ranges.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment 1: Toxicity was observed at 158.1μg/plate and above in all strains in the absence of S9. In the presence of S9, evidence of toxicity was observed in strains TA 100 and TA 1537 at 158.1 μg/plate and above and in strains TA 98, TA 1535 and TA 102 at 500 μg/plate and above.
- Experiment 2: Toxicity was observed at 160 μg/plate and above in all strains in the absence and in the presence of S9. In addition to this, evidence of toxicity was also observed at 25.6 and 64 μg/plate in strain TA100 in the presence of S9 and at 64 μg/plate in strains TA 98, TA 1535 and TA 1537 in the presence of S9 and strain TA 102 in the absence of S9.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the test conditions, Terpinolene multiconstituent is not considered as mutagenic in S. typhimurium strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102).
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, TA 100 and TA 102) were exposed to Terpinolene multiconstituent at the following concentrations:

- Experiment 1: 5, 15.81, 50, 158.1, 500, 1581 and 5000 μg/plate, with and without S9 mix in all 5 strains (plate incorporation method)

- Experiment 2: 4.096, 10.24, 25.6, 64, 160 and 400 μg/plate, with S9 mix (preincubation method) and without S9 mix (plate incorporation method) in all 5 strains [1.638 μg/plate employed for treatment of all strains in the absence of S9 and strains TA 100 and TA 1537 in the presence of S9 only; 1000 μg/plate employed for treatment of strains TA 98, TA 1535 and TA 102 in the presence of S9 only.]

Metabolic activation system used in this test was10 % S9 mix. S9 fraction was prepared from liver homogenates of male Sprague Dawley rats induced with Aroclor 1254. Vehicle control and positive control groups were also included in mutagenicity tests.

In Experiment 1, toxicity was observed at 158.1 μg/plate and above in all strains in the absence of S9. In the presence of S9, evidence of toxicity was observed in strains TA 100 and TA 1537 at 158.1 μg/plate and above and in strains TA 98, TA 1535 and TA 102 at 500 μg/plate and above. In Experiment 2, toxicity was observed at 160 μg/plate and above in all strains in the absence and in the presence of S9. In addition to this, evidence of toxicity was also observed at 25.6 and 64 μg/plate in strain TA 100 in the presence of S9 and at 64 μg/plate in strains TA 98, TA 1535 and TA 1537 in the presence of S9 and strain TA 102 in the absence of S9. The positive and vehicle controls induced the appropriate responses in the corresponding strains. Test item did not induce statistically significant increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of metabolic activation.

Under the test conditions, Terpinolene multiconstituent is not considered as mutagenic in this bacterial system.

Categories Display