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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From April 16 to May 24, 2010
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:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Delta-3-Carene
- Physical state: Pale slightly amber colour liquid
- Analytical purity: 94.8%
- Impurities (identity and concentrations): Myrcene 1.3%; Dipentene 0.9%; Beta Pinene 0.8%
- Purity test date: 1 April 2010
- Lot/batch No.: EC505R
- Date received: 8 April 2010
- Expiration date of the lot/batch: 31 March 2011
- Storage condition of test material: 2-8 ºC under nitrogen in the dark

Method

Target gene:
Not applicable
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium 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:
Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9), obtained from Molecular Toxicology Incorporated, USA
Test concentrations with justification for top dose:
- Range-finder experiment: 1.6, 8, 40, 200, 1000 and 5000 µg/plate (with and without S-9);
- Experiment 1: 0.064, 0.32, 1.6, 8, 40, 200 and 1000 µg/plate, (with and without S-9);
- Experiment 2: 7.813, 15.63, 31.25, 62.5, 125, 250 and 500 µg/plate (strain TA102 in the absence of S-9 and strains TA 100 and TA 1535 in the presence of S-9);
0.9766, 1.953, 3.906, 7.813, 15.63, 31.25 and 62.5 µg/plate (strains TA 98, TA 100, TA 1535 and TA 1537 in the absence of S-9 and strains TA 98, TA 1537 and TA 102 in the presence of S-9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: Solubility of delta-3-carene in DMSO = 100 mg/mL
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See table 1
Details on test system and experimental conditions:
METHOD OF APPLICATION: Plate incorporation method; as the results of Experiment 1 were negative, treatments in the presence of S-9 in experiment 2 included a pre-incubation step (incubation for 1 hour at 37±1°C).
Due to the suspected volatility of the test item, test article plates were incubated in sealed boxes with each concentration of test article in a separate box and the vehicle and positive controls were incubated in a separate incubator.
In the Range-Finder experiment, the vehicle and positive control plates were not incubated in a separate incubator to the test article plates in error. These data were used for toxicity assessment only.
In Experiment 1, the plates were not incubated in sealed boxes in error and were placed in the same incubator as the control plates. The controls are consistent with the historical control data and the toxicity profile is the same as that in the Range-Finder where the treatment plates for each concentration were placed in a sealed box. The suspected volatility of the test article has not affected the other plates and is considered not to have impacted on the integrity of the study.

DURATION
For all assays, bacteria were cultured at 37±1°C for 10 hours in nutrient broth, containing ampicillin (TA98, TA100) or ampicillin and tetracycline (TA102) as appropriate. Incubation was carried out with shaking in an anhydric incubator. All treatments were completed within 6 hours of the end of the incubation period.
After plating with test substance or control, the plates were inverted and incubated at 37±1°C in the dark for 3 days.

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS:
Range-finding test: triplicate plates; negative (vehicle) and positive controls were included in quintuplicate and triplicate, respectively.
Main experiments: triplicate plates; negative (vehicle) controls were included in quintuplicate, and positive controls were included in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants; thinning of background bacterial lawn.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic if:
- Dunnett's test gave a significant response (p=< 0.01) which was concentration related
- The positive trend/effects described above were reproducible.
Negative: If all of the above criteria were not 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.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see additionnal information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
see additionnal information on results
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- Range-finder experiment: slight thinning of the background bacterial lawn was observed at 40 µg/plate and above with and without S-9

- Experiment 1: diminution of the background bacterial lawn: At 40 µg/plate and above in strains TA98, TA100, TA1535 and TA1537 in the absence of S-9 and strains TA98, TA1537 and TA102 in the presence of S-9; at 200 µg/plate and above in strain TA102 in the absence of S-9 and strains TA100 and TA1535 in the presence of S 9.
Reduction in revertant numbers: in strain TA1537 in the absence of S-9 at 200 µg/plate and above and in strains TA1537 and TA102 in the presence of S-9 at 1000 µg/plate

- Experiment 2: slight thinning of the background bacterial lawn: at 31.25 µg/plate and above in strains TA98, TA100, TA1535 and TA1537 in the absence of S-9 and strains TA98, TA537 and TA102 in the presence of S-9 and at 250 µg/plate and above in strain TA102 in the absence of S-9 and strains TA100 and TA1535 in the presence of S-9
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, delta-3-carene is not considered as mutagenic in this bacterial system according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation(EC) N° 1272/2008. 
Executive summary:

In a GLP study performed according to OECD guideline 471, delta-3 -carene was tested for mutagenicity using Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 with the plate incorporation and preincubation methods in the presence and absence of metabolic activation system (S-9 mix).

Based on the results of range-finder test, using TA100 tested at concentrations between 1.6 and 5000 µg/plate with and without S-9, all strains in experiment 1 were tested with and without S-9 at the concentration range of 0.064 - 1000 µg/plate. Due to the cytotoxicity found in experiment 1, the following concentration ranges were tested in the second experiment, using the preincubation method (1h at 37°C) when S-9 was added: 7.813 - 500 µg/plate (strain TA102 in the absence of S-9 and strains TA100 and TA1535 in the presence of S-9) or 0.9766 - 62.5 µg/plate (strains TA98, TA100, TA1535 and TA1537 in the absence of S‑9 and strains TA98, TA1537 and TA102 in the presence of S-9).

In range-finder experiment, evidence of toxicity was observed at 40 µg/plate and above in the absence and presence of S-9. In experiment 1, evidence of toxicity was observed at 40 µg/plate and above in strains TA98, TA100, TA1535 and TA1537 in the absence of S‑9 and strains TA98, TA1537 and TA102 in the presence of S‑9 and at 200 µg/plate and above in strain TA102 in the absence of S-9 and strains TA100 and TA1535 in the presence of S-9. In experiment 2, toxicity was observed at 31.25 µg/plate and above in strains TA98, TA100, TA1535 and TA1537 in the absence of S-9 and strains TA98, TA1537 and TA102 in the presence of S-9 and at 250 µg/plate and above in strain TA102 in the absence of S-9 and strains TA100 and TA1535 in the presence of S-9. No precipitation of test substance was observed at any of the doses used.

The positive controls induced the appropriate responses in the corresponding strains. Delta-3-carene showed no substantial increases in revertant colony numbers over control count obtained with any of the tester strains at any concentrations in either presence or absence of S-9.

Under the test conditions, delta-3-carene is not considered as mutagenic in this bacterial system according to the criteria of the Annex VI to the Directive 67/548/EEC and CLP Regulation(EC) N° 1272 /2008.