Registration Dossier
Registration Dossier
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EC number: 233-986-8 | CAS number: 10482-56-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. In several Ames assays for mutagenecity on Salmonella typhimurium strains TA 97a, TA 98, TA 100, TA 1535 TA 1537, TA 1538 or TA 102 and Escherichia coli strain WP2 uvrA, alpha terpineol and terpineol were found not mutagenic with and without metabolic activation. Only terpineol was found weakly mutagenic in TA102 tester strain in one single study. Based on these results, the read-across approach was applied and laevo alpha terpineol can be considered to be not mutagenic in bacteria with and without metabolic activation.
In vitro cytogenicity in mammalian cells. Weight of Evidence: Read-across approach. Based on the read-across approach from the analogue terpineol tested in an in vitro chromosome aberration study with CHL/IU cells in presence and absence of metabolic activation, laevo alpha terpineol is not considered to show clastogenic potential.
In vitro gene mutation study in mammalian cells. Weight of Evidence: Read-across approach. Based on the read-across approach from the analogue alpha terpineol found negative in the Mouse Lymphoma Assay in the presense and in the absence of metabolic activation, laevo alpha terpienol can be considered not to be mutagenic in mammalian cells.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 20 September 2012 – 22 March 2013
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine-requiring gene in Salmonella typhimurium and tryptophan-requiring gene in Escherichia coli
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 fraction prepared from male Sprague-Dawley rat liver and induced by Phenobarbital (PB) and 5,6-benzoflavone (BF)
- Test concentrations with justification for top dose:
- Preliminary experiment: 19.5, 78.1, 313, 1250 and 5000 μg/plate.
In the preliminary test, growth inhibition was observed at 1250 μg/plate for all strains without metabolic activity. When tested with metabolic activity, growth inhibition was observed at 313 μg/plate for strains TA 98, TA 1535 and TA 1537, and at 1250 μg/plate for strains TA 100 and E. Coli.
The highest dose selected for the main experiments for each strain was the lowest dose showing growth inhibition in the preliminary test. This dose was further diluted 5 times with the ratio of 2 to obtain 6 doses.
Main tests 1 and 2 (all strains -S9 and TA 98, TA 1535 and TA 1537 +S9): 39.1, 78.1, 156, 313, 625 and 1250 μg/plate
Main tests 1 and 2 (TA 100 and E. Coli +S9): 9.77, 19.5, 39.1, 78.1, 156 and 313μg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to solubility in water of 1.98 g/L, a solubility test was performed on DMSO. As a result, the test substance was dissolved in DMSO at 50 mg/mL and no reactivity such as generation of heat or gas was observed, so the test was conducted using DMSO as a solvent. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- benzo(a)pyrene
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2); 2-Methoxy-6-chloro-9-[ 3-(2-chloro ethyl)-aminopropylamino] acridine 2HCL (ICR-191); 2-Aminoanthracene (2AA)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation: 0.1 mL of solvent or positive control or test solution, 0.5 mL of S9 mix or 0.1 M phosphate buffer solution (pH 7.4) and 0.1 mL of culture solution of each strain were added to the test tube. Preincubation was carried out with shaking (80 rpm) for 20 minutes at 37ºC. After completion of the preincubation 2.0 mL of top agar were added and after stirring the mixture was uniformly layered on a minimum glucose agar plate medium. Once confirmed that the top agar layered on the minimum glucose agar plate medium was solidified, the plate was turned upside down, placed in the incubator, and incubate at 37ºC for 49 hours in preliminary test and for 48.5 h in the main tests.
- Cell density at seeding (if applicable): 1 x 10^9 cells/mL.
DURATION
- Preincubation period: 20 min.
- Exposure duration: 48.5 h
SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants (revertant bacteria) can grow due to their capability to synthesize the essential amino acid.
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition. The presence or absence of growth inhibition was observed using a stereoscopic microscope. - Evaluation criteria:
- A result was considered positive when the number of revertant colonies in the test substance treated group showed an increase of 2 times or more as compared with the number of spontaneous revertant colonies (negative control value) and a dose response and reproducibility were observed. Also, a positive result was decided when a clear dose response was not shown but an increase of more than twice the number of spontaneously reversed mutant colonies was counted and when reproducibility was confirmed in two main tests.
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 625 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 625 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 313 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 1250 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 625 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 313 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 625 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 313 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Conclusions:
- Terpineol did not show any mutagenic effect in bacteria under test conditions with and without metabolic activation.
- Executive summary:
Terpineol was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, 1535 and TA1537 and Escherichia coli strain WP2 uvrA with and without metabolic activation (S9). The experiment was performed according to OECD guideline 471 with GLP. Based on growth inhibition examined in a preliminary test up to a maximum dose of 5000μg/plate, tested concentrations in the main test were: 39.1, 78.1, 156, 313, 625 and 1250μg/plate for all strains without metabolic activationand for strains TA 98, TA 1535 and TA 1537 with metabolic activation, and 9.77, 19.5,39.1, 78.1, 156 and 313μg/plate for strains TA 100 and E. Coli WP2 with metabolic activation. Two main tests were performed in triplicate using preincubation method and DMSO as solvent. Negative and positive controls were within background data of the test laboratory. Under test conditions, terpineol was found not mutagenic in all strains tested with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine-requiring gene in Salmonella typhimurium
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- other: uvrB and rfa mutated
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- other: uvrB and rfa mutated
- Species / strain / cell type:
- S. typhimurium, other: TA97a
- Additional strain / cell type characteristics:
- other: uvrB and rfa mutated
- Species / strain / cell type:
- S. typhimurium TA 102
- Additional strain / cell type characteristics:
- other: uvrB and rfa mutated
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 fraction induced by Aroclor 1254
- Test concentrations with justification for top dose:
- Preliminary test carried out with TA100 strain without and with addition of S9 mixture: 0, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 2000, 2500, 2750 and 3000 μg/plate
Main assay (upper limit of the dose interval tested was either the highest non-toxic dose or the lowest toxic dose determined in the preliminary assay): 0, 25, 50,100, 250, 500, 750, 1000, 1250, 1500, 2000 and 2500 μg/plate. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- sodium azide
- benzo(a)pyrene
- mitomycin C
- other: 2-aminoanthracene (TA100/+S9 (1 µg/plate); TA98/+S9 (0.5 µg/plate)); nitro-o-phenilene-diamine (TA98/-S9 (1 µg/plate)); 2-Aminofluorene (TA97a/+S9 (10 µg/plate))
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Direct plate incorporation method: 2 ml of top-agar was mixed with 100 µl of an overnight grown culture of S. typhimurium, 100 µl of the test substance (diluted in ethanol analytical grade, Merck, KGaA), the negative control, or the positive control (PC) and 500 µl of the phosphate buffer or the S9 mixture. Plates were incubated at 37ºC for 72h in the dark and then scored for revertant his+ bacteria colonies. Each determination was made in triplicate and two independent experiments were carried out.
- Cell density at seeding: For all assays, an inoculum (200 μl) of a thawed permanent culture was added to 20ml of Nutrient Broth No. 2 and incubated at 37ºC with shaking until a concentration of approximately 1–2 x10^9 bacteria per millilitre was obtained.
DURATION
- Exposure duration: 72h
SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
Toxicity of the test compound to S. typhimurium strain TA100 was investigated in the preliminary assay. Toxicity was apparent either as a reduction in the number of his+ revertant bacteria colonies and or as a change of auxotrophic background growth (i.e. the background lawn).
Doses at which toxicity appeared as an alteration of the background lawn are marked with an asterisk in tables 1 and 2 (Any other information on results incl. Tables).
OTHER:
-Lyophilized rat liver S9 fraction induced by Aroclor 1254 was purchased from Moltox (Molecular Toxicology, Annapolis, USA). The S9 mixture was prepared as follows: 7.0ml of ultrapure water; 10.5ml of 200mM sodium phosphate buffer pH7.4; 0.84ml of 100mM NADP solution; 0.105ml of 1M glucose-6-phosphate; 0.42ml of 1.65M KCl + 0.4M MgCl2 salt solution; and 2.1ml of lyophilized S9 fraction reconstituted with distilled water. - Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- (up to highest dose tested of 2000 μg/plate)
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 2000 μg/plate)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Remarks:
- (up to highest dose tested of 2500 μg/plate)
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 2500 μg/plate)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- (up to highest dose tested of 1500 μg/plate)
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Remarks:
- up to highest dose tested of 2500 μg/plate
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium, other: TA97a
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- (up to highest dose tested of 1500 μg/plate)
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium, other: TA97a
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Remarks:
- (up to highest dose tested of 2000 μg/plate)
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 2000 μg/plate)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Terpineol was found to be not mutagenic in TA 100, TA 98 and TA 97a tester strains and weakly mutagenic in TA102 tester strain with and without metabolic activation.
- Executive summary:
Terpineol was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, TA97a and TA102 with and without metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. Every determination was made in triplicate and each experiment was repeated once in order to check the reproducibility of the results. In contrast to the absence of mutagenicity towards TA97a, TA98 and TA100 strains, terpineol produced a dose-related increase in the number of TA102 revertants. The maximum effects were a 2.0-fold increase in the number TA102 revertants at doses as high as 750 µg/plate in the absence of S9 mixture, and a 2.2-fold increase at doses as high as 1250 µg/plate in the presence of S9 mixture. The negative as well as the positive results were reproduced by a confirmatory experiment. In conclusion, terpineol was found to be not mutagenic in TA 100, TA 98 and TA 97a strains but weakly mutagenic in TA102 strain with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- (only one replicate was conducted)
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine-requiring gene in Salmonella typhimurium
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: uvrB and rfa mutated
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S9 fraction induced by Aroclor 1254 (S-9A)
- Test concentrations with justification for top dose:
- 3 μmol/plate (463 μg/plate)
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (+S9); N-methyl-N'-nitro-N-nitrosoguanidin (-S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Cultures were grown in Oxoid nutrient broth No. 2. Revertants were scored on glucosenminimal salts medium supplemented with 0.05 μmol histidine and 0.05 μmol biotin. Plates used for viable counts contained 10 μmol histidine (and 0.05 μmol biotin). The experiments were carried out essentially as described by Ames.
SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.
NUMBER OF REPLICATIONS: 1
DETERMINATION OF CYTOTOXICITY
Toxicity was determined based on the absence of a background lawn of bacteria on the plates. If absence of a background lawn was found the test was repeated with a lower concentration of the substance. - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- at 3 μmol/plate
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- at 3 μmol/plate
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- at 3 μmol/plate
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- at 3 μmol/plate
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: rat liver S9 fraction induced by Aroclor 1254 (S-9A)
- Conclusions:
- Alpha terpineol was not mutagenic in all strains tested with and without metabolic activation.
- Executive summary:
Alpha terpineol was tested using the Ames assay for mutagenecity on Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation (S9). The test item was tested at 3 μmol/plate on strains TA 98, TA 100, TA 1535 and TA 1537 both with and without metabolic activation using a liver fraction (S-9) from Aroclor 1254. Under these test conditions, alpha terpineol was found not mutagenic in all strains tested with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- documentation insufficient for assessment
- Remarks:
- Data published in a peer reviewed journal. Original study report not available.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- not specified
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine-requiring gene in Salmonella typhimurium
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- other: uvrB and rfa mutated
- Species / strain / cell type:
- S. typhimurium TA 1538
- Additional strain / cell type characteristics:
- other: uvrB and rfa mutated
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor 1254-induced rat liver S9
- Test concentrations with justification for top dose:
- 10000 μg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: no data
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- not specified
- True negative controls:
- not specified
- Positive controls:
- not specified
- Details on test system and experimental conditions:
- DURATION
- Exposure duration: 48h
SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid. - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Conclusions:
- Alpha terpineol was not mutagenic in all strains tested with and without metabolic activation.
- Executive summary:
Alpha terpineol was tested for mutagenecity on Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. Alpha terpineol was not mutagenic in all strains tested with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine-requiring gene in Salmonella typhimurium
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- Liver S9 homogenate prepared from male Sprague-Dawley rats and Syrian golden hamsters induced with Aroclor 1254.
- Test concentrations with justification for top dose:
- 0, 10, 33, 100, 333 and 1000 μg/plate.
These doses were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels, one plate per dose, were tested in both the presence and the absence of induced hamster S9. As some toxicity was observed at highest doses, a total maximum dose of 1 mg per plate was used. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- not specified
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Direct plate incorporation method: For testing in the absence of S9 mix, 100 μL of the tester strain and 50 μL of the solvent or test chemical were added to 2.5 mL of molten selective top agar at 45 ± 2 ºC. When S9 was used, 0.5 mL of S9 mix, 50 μL of tester strain, and 50 μL of solvent or test chemical were added to 2.0 mL of molten selective top agar at 45 ± 2 ºC. After it was vortexed, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. After the overlay had solidified, the plates were incubated for 48 h at 37 ± 2 ºC.
- Cell density at seeding: Cultures were grown overnight in Oxoid nutrient broth no. 2 and were removed from incubation when they reached a density of 1-2x10e9 cells/mL.
DURATION
- Exposure duration: 48h
SELECTION AGENT (mutation assays): the lack of amino-acid in the medium. Only the mutants can grow due to their capability to synthesize the essential amino acid.
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
A preliminary dose range-finding study using strain TA100 (both with and without metabolic activation) was performed. - Evaluation criteria:
- For the test substance to be considered positive, it had to induce at least a doubling (TA98, TA100, and TA1535) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test substance. If the study showed a dose response with a less than 3-fold increase on TA1537 or TA1538, the response had to be confirmed in a repeat experiment.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence
and the absence of induced hamster S9. As some toxicity was observed at the highest doses, a total maximum dose of 1 mg of test chemical per plate was used in the main study. - Conclusions:
- alpha terpineol was not mutagenic in all strains tested with and without metabolic activation.
- Executive summary:
Alpha terpineol was tested for mutagenecity on Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 with and without metabolic activation (S9 mix prepared from both rat and hamster liver). The experiment was performed using the Ames Salmonella assay for mutagenicity. In a preliminary dose range-finding study ten dose levels of the test substance, one plate per dose, were tested in both the presence and the absence of induced hamster S9 using strain TA100. As some toxicity was observed, a maximum dose of 1 mg per plate was used. Based on this preliminary study, the selected doses for the main study were 0, 10, 33, 100, 333 and 1000 μg/plate. DMSO was used as solvent and tested as solvent control. Under these experimental conditions, the test substance was found not mutagenic in all strains tested with and without metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 4 October, 2012 - 22 March, 2013
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- mammalian cell line, other: Chinese Hamster Lung Fibroblast (CHL /IU)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Human Science Research Resource Bank
- Suitability of cells: Properties of cryopreserved cells were periodically checked (culture mode, cell doubling time within 15 hours 20 hours, average number of chromosomes, contamination of mycoplasma, etc.).
- Cell cycle length, doubling time or proliferation index: cell doubling time within 15 to 20 hours.
- Number of passages if applicable: The cell passage number used was 19 passages in the cell proliferation inhibition test, 25 passages in the short treatment method for the chromosomal aberration test, and 8 passages in the continuous treatment method.
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimum Essential Medium (MEM) supplemented with 10% (v/v) heated inactivated bovine serum. Using a carbonic acid gas incubator, cultivation was carried out under conditions of 5% CO2, 37ºC and high humidity. Passage was done every 1-4 days.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes. - Metabolic activation:
- with and without
- Metabolic activation system:
- Co-factor-supplemented S9 from Sprague-Dawley male rats induced with a combination of phenobarbital (PB) and 5, 6-benzoflavone (BF)
- Test concentrations with justification for top dose:
- Cell growth inhibition test: 1600, 800, 400, 200, 100, 50.0, 25.0, 12.5 and 0 (negative) μg/mL.
Chromosome aberration test:
short-time treatment (+S9): 500, 400, 300, 200, 100 and 0 (negative) μg/mL
short-time treatment (-S9) and continuous treatment (24 h and 48 h): 400, 300, 200, 100 and 0 (negative) μg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in culture medium.
- Cell density at seeding (if applicable): 2 x 10^4 cells per plate
DURATION
- Exposure duration: 6 h (short time treatment); 24 h and 48 h (continuous treatment)
SPINDLE INHIBITOR (cytogenetic assays): 0.1 mL of colcemid (10 μg / mL solution) was added about 2 hours before the end of the culture for preparing specimens for chromosome observation.
STAIN (for cytogenetic assays): 2% Giemsa solution for about 15 minutes
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: After treatment, cells were incubated with 0.25% trypsin, suspended in 0.075 M KCl and incubated for about 15 minutes. Cells were then centrifuged and fixed with methyl alcohol: Acetic acid = 3:1 solution. Finally, the cells were stained with 2% Giemsa solution for about 15 minutes to prepare chromosome specimens.
NUMBER OF CELLS EVALUATED: 200 cells per group
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200 cells analysed per group.
DETERMINATION OF CYTOTOXICITY
- Method: cell growth inhibition test
This assay was conducted in duplicate for short-term treatment group (6 h) with and without metabolic activation and for continuous treatment group (24 h and 48 h) without metabolic activation at doses of 1600, 800, 400, 200, 100, 50.0, 25.0, 12.5 and 0 (negative control) μg/mL. The cell-growth ratio was determined against the negative control regarded as 100% growth. Condition of cells was observed at the end of treatment. Color of medium was observed immediately after addition of the test solutions. Precipitates/crystals were observed immediately after addition of the test solutions and at the end of treatment. The 50% cell proliferation inhibiting concentration of the test substance was determined for every treatment group.
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes - Evaluation criteria:
- The evaluation of results was performed according to the following criteria:
If incidence of chromosomal aberrations is less than 5% the test is considered negative
If incidence of chromosomal aberrations is more than 5% and less than 10% the test is considered as false positive.
If incidence of chromosomal aberrations is more than 10% the test is considered positive.
For the overall assessment the structural chromosomal aberrations excluding gaps were used. Also, a dose dependency or reproducibility was considered for the final assessment. - Key result
- Species / strain:
- mammalian cell line, other: Chinese Hamster Lung Fibroblast (CHL /IU)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- see tables 1-4 on "Any other information on results incl. tables"
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Immediately after addition of the test solution, precipitation was observed at doses of 400 μg/mL or more in all treatment groups. At the end of treatment, precipitation was observed at 500 μg/mL(+S9) and 400 μg/mL(-S9) in the short-time treatment group. In the continuous treatment group no precipitation was observed at the end of treatment.
- Other confounding effects: No change in color tone of the culture solution was observed after addition of the test solution.
RANGE-FINDING/SCREENING STUDIES:
In the preliminary study, immediately after addition of the test solution, precipitation was observed at doses of 400 μg/mL or more in all treatment methods. No change in color tone of the culture solution was observed.
At the end of the treatment, precipitation was observed at a dose of 400 μg mL or more for metabolic activation in the short-time treatment group and at a dose of 800 μg/mL or more by non-metabolic activation and continuous treatment group. The 50% cell-growth inhibition concentration (approximate value) was calculated to be 449 μg/mL in the short-time treatment group (+S9), 295 μg/mL in the short-time treatment group (-S9), 276 μg/mL in the 24-hour continuous treatment group and 242 μg/mL in the 48-hour continuous treatment group.
- Conclusions:
- In an in vitro chromosome aberration study, terpineol was found not clastogenic when tested in CHL/IU cells in vitro with or without metabolic activation.
- Executive summary:
Terpineol was tested in an in vitro chromosome aberration study with cultured Chinese hamster lung CHL/IU cells in presence and absence of metabolic activation using a method according to OECD guideline 473. Based on a preliminary cytotoxicity study consisting on growth inhibition the tested concentrations were 500, 400, 300, 200, 100 and 0 (negative) μg/mL in the short-time treatment (+S9) and 400, 300, 200, 100 and 0 (negative) μg/mL in the short-time treatment (-S9) and continuous treatment (24 h and 48 h). Duplicate cultures were exposed to the test compound for 6 h (short time treatment) and for 24 h and 48 h (continuous treatment) before sampling. There were no statistically significant or dosage-related increases in both structural and numerical chromosome aberrations compared with the concurrent control (DMSO), with and without metabolic activation at both short-term and continuous treatment. Both positive controls, cyclophosphamide with metabolic activation and mitomycin C without metabolic activation, produced highly significant increases in the numbers of aberrant cells compared to control. From the above results, it was concluded that terpineol does not induce chromosome structural abnormality and chromosome number abnormality under the test conditions.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Deviations:
- not specified
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- Thymidine Kinase Gene
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: Dr. Donald Clive, Burroughs Wellcome Co. (Research Triangle Park, NC).
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Fischer’s medium for leukemic cells of mice (Gibco, Grand Island, NY, or Quality Biological, Gaithersburg, MD) supplemented with 10% horse serum (Gibco or Hyclone, Logan, UT) and 0.02% pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI). - Metabolic activation:
- with and without
- Metabolic activation system:
- liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats
- Test concentrations with justification for top dose:
- - S9 mix: 0.14, 0.2, 0.26, 0.32 and 0.38 µL/mL
+S9 mix: 0.17, 0.27, 0.36, 0.46 and 0.56 µL/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not specified. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- ( DMSO)
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium.
- Cell density at seeding (if applicable): Cells in the cultures were adjusted to 3x10e5/mL at 24 h intervals. They were then cloned (1x10e6 cells/plate for mutant selection and 200 cells/plate for viable count determinations) in soft agar medium.
DURATION
- Exposure duration: 4h
- Expression time (cells in growth medium, to allow expression of the TK- mutation ): 48h
- Selection time (plating for -trifluorothymidine (TFT) resistance): 10-12 days
SELECTION AGENT (mutation assays): trifluorothymidine (TFT) (final concentration, 3 µg /mL)
NUMBER OF REPLICATIONS: Duplicate cultures were used for each treatment.
NUMBER OF CELLS EVALUATED: 1.2 x 10e7 cells
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity: The toxicity was determined both with and without liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats. Cells at a concentration of 6 x 10e5/mL (6 x10e6 cells total) were exposed for 4 h to a range of concentrations from 0.0005 to 10000 µg/mL. The cells were then washed, resuspended in growth medium, and incubated at 37 ± 1ºC for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent control. The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity. - Evaluation criteria:
- Mutant frequencies were expressed as mutants per 10e6 surviving cells.
A positive result was interpreted following two different methods:
1. Using a doubling of the mutant frequency over the concurrent solvent-treated control value, together with evidence of a dose-related increase (cited as old evaluation in the report).
2. if a concentration-related increase in mutant frequency is observed and one or more dose levels with 10% or greater total growth exhibit mutant frequencies of ≥100 mutants per 10e6 clonable cells over the background level (cited as new evaluation in the report). - Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 27-157% RTG
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 19-112% RTG
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Conclusions:
- The test substance was found negative in the Mouse Lymphoma Assay in the presense and in the absence of metabolic activation.
- Executive summary:
An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus (OECD test guideline 490) to test the potential to cause gene mutation. Treatment was performed for 4 hours with and without metabolic activation (±S9 mix). DMSO was used as solvent. The test item was examined previously in a cytotoxicity Test. Based on these results, the test item concentrations in the mutation assay ranged from 0.14 to 0.56 µL/mL. The experiments were performed using appropriate negative (vehicle) and positive control samples. Under conditions of the assay, the test substance was found negative in the presense and in the absence of metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance alpha terpineol which shares the same functional groups with the substance L-alpha terpineol also has comparable values for the relevant molecular properties.
See attached the reporting format - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- at 3 μmol/plate
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- at 3 μmol/plate
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- at 3 μmol/plate
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Remarks:
- at 3 μmol/plate
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in this strain of bacteria
- Conclusions:
- Based on the read-across approach from the analogue alpha terpineol, laevo alpha terpineol is considered to be not mutagenic in tester strains TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation.
- Executive summary:
Alpha terpineol was tested using the Ames assay for mutagenecity on Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation (S9). The test item was tested at 3 μmol/plate on strains TA 98, TA 100, TA 1535 and TA 1537 both with and without metabolic activation using a liver fraction (S-9) from Aroclor 1254. Under these test conditions, alpha terpineol was found not mutagenic in all strains tested with and without metabolic activation. Based on these results, the read-across approach was applied and laevo alpha terpineol is considered to be not mutagenic in tester strains TA 98, TA 100, TA 1535 and TA 1537 with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance terpineol which shares the same functional groups with the substance L-alpha terpineol also has comparable values for the relevant molecular properties.
See attached the reporting format - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- (up to highest dose tested of 2000 μg/plate)
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 2000 μg/plate)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Remarks:
- (up to highest dose tested of 2500 μg/plate)
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 2500 μg/plate)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- (up to highest dose tested of 1500 μg/plate)
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Remarks:
- up to highest dose tested of 2500 μg/plate
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium, other: TA97a
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Remarks:
- (up to highest dose tested of 1500 μg/plate)
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium, other: TA97a
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Remarks:
- (up to highest dose tested of 2000 μg/plate)
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 2000 μg/plate)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in this strain of bacteria
- Conclusions:
- Based on the read-across approach from the analogue terpineol, laevo alpha terpineol is determined to be not mutagenic in TA 100, TA 98 and TA 97a tester strains and weakly mutagenic in TA102 tester strain with and without metabolic activation.
- Executive summary:
Terpineol was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, TA97a and TA102 with and without metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. Every determination was made in triplicate and each experiment was repeated once in order to check the reproducibility of the results. In contrast to the absence of mutagenicity towards TA97a, TA98 and TA100 strains, terpineol produced a dose-related increase in the number of TA102 revertants. The maximum effects were a 2.0-fold increase in the number TA102 revertants at doses as high as 750 µg/plate in the absence of S9 mixture, and a 2.2-fold increase at doses as high as 1250 µg/plate in the presence of S9 mixture. The negative as well as the positive results were reproduced by a confirmatory experiment. In conclusion, terpineol was found to be not mutagenic in TA 100, TA 98 and TA 97a strains but weakly mutagenic in TA102 strain with and without metabolic activation. Based on these results, the read-across approach was applied and laevo alpha terpineol is determined to be not mutagenic in TA 100, TA 98 and TA 97a tester strains and weakly mutagenic in TA102 tester strain with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance alpha terpineol which shares the same functional groups with the substance L-alpha terpineol also has comparable values for the relevant molecular properties.
See attached the reporting format - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in this strain of bacteria
- Conclusions:
- Based on the read-across approach from the analogue alpha terpineol, laevo alpha terpineol is considered to be not mutagenic in tester strains TA1535, TA1537, TA1538, TA98 and TA100 with and without metabolic activation.
- Executive summary:
Alpha terpineol was tested for mutagenecity on Salmonella typhimurium strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation (S9). The experiment was performed using the Ames Salmonella assay for mutagenicity. Alpha terpineol was not mutagenic in all strains tested with and without metabolic activation. Based on these results, the read-across approach was applied and laevo alpha terpineol is considered to be not mutagenic in tester strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance alpha terpineol which shares the same functional groups with the substance L-alpha terpineol also has comparable values for the relevant molecular properties.
See attached the reporting format - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not valid
- Positive controls validity:
- valid
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in this strain of bacteria
- Conclusions:
- Based on the read-across approach from the analogue alpha terpineol, laevo alpha terpineol is considered to be not mutagenic in tester strains TA1535, TA1537, TA1538, TA98 and TA100 with and without metabolic activation.
- Executive summary:
Alpha terpineol was tested for mutagenecity on Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 with and without metabolic activation (S9 mix prepared from both rat and hamster liver). The experiment was performed using the Ames Salmonella assay for mutagenicity. In a preliminary dose range-finding study ten dose levels of the test substance, one plate per dose, were tested in both the presence and the absence of induced hamster S9 using strain TA100. As some toxicity was observed, a maximum dose of 1 mg per plate was used. Based on this preliminary study, the selected doses for the main study were 0, 10, 33, 100, 333 and 1000 μg/plate. DMSO was used as solvent and tested as solvent control. Under these experimental conditions, the test substance was found not mutagenic in all strains tested with and without metabolic activation. Based on these results, the read-across approach was applied and laevo alpha terpineol is considered to be not mutagenic in tester strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100 with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance terpineol which shares the same functional groups with the substance L-alpha terpineol also has comparable values for the relevant molecular properties.
See attached the reporting format. - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 625 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 625 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 313 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 1250 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 625 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 313 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 625 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 313 μg/plate and above)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in this strain of bacteria
- Conclusions:
- Based on the read-across approach from the analogue terpineol, laevo alpha terpineol is considered to be not mutagenic in bacteria with and without metabolic activation.
- Executive summary:
Terpineol was tested for mutagenecity on Salmonella typhimurium strains TA100, TA98, 1535 and TA1537 and Escherichia coli strain WP2 uvrA with and without metabolic activation (S9). The experiment was performed according to OECD guideline 471 with GLP. Based on growth inhibition examined in a preliminary test up to a maximum dose of 5000μg/plate, tested concentrations in the main test were: 39.1, 78.1, 156, 313, 625 and 1250μg/plate for all strains without metabolic activationand for strains TA 98, TA 1535 and TA 1537 with metabolic activation, and 9.77, 19.5,39.1, 78.1, 156 and 313μg/plate for strains TA 100 and E. Coli WP2 with metabolic activation. Two main tests were performed in triplicate using preincubation method and DMSO as solvent. Negative and positive controls were within background data of the test laboratory. Under test conditions, terpineol was found not mutagenic in all strains tested with and without metabolic activation. Based on these results, the read-across approach was applied and laevo alpha terpineol is considered to be not mutagenic in tester strains TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 uvrA, with and without metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance terpineol which shares the same functional groups with the substance L-alpha terpineol also has comparable values for the relevant molecular properties.
See attached the reporting format - Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- mammalian cell line, other: Chinese Hamster Lung Fibroblast (CHL /IU)
- 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
- Remarks on result:
- other: read-across from an analogue determined not to induce chromosome abnormalities.
- Conclusions:
- Based on the read-across approach from the analogue terpineol tested in an in vitro chromosome aberration study with CHL/IU cells in presence and absence of metabolic activation, laevo alpha terpineol is not considered to show clastogenic potential.
- Executive summary:
Terpineol was tested in an in vitro chromosome aberration study with cultured Chinese hamster lung CHL/IU cells in presence and absence of metabolic activation using a method according to OECD guideline 473. Based on a preliminary cytotoxicity study consisting on growth inhibition the tested concentrations were 500, 400, 300, 200, 100 and 0 (negative) μg/mL in the short-time treatment (+S9) and 400, 300, 200, 100 and 0 (negative) μg/mL in the short-time treatment (-S9) and continuous treatment (24 h and 48 h). Duplicate cultures were exposed to the test compound for 6 h (short time treatment) and for 24 h and 48 h (continuous treatment) before sampling. There were no statistically significant or dosage-related increases in both structural and numerical chromosome aberrations compared with the concurrent control (DMSO), with and without metabolic activation at both short-term and continuous treatment. Both positive controls, cyclophosphamide with metabolic activation and mitomycin C without metabolic activation, produced highly significant increases in the numbers of aberrant cells compared to control. From the above results, it was concluded that terpineol does not induce chromosome structural abnormality and chromosome number abnormality under the test conditions. Based on these results, the read-across approach was applied and laevo alpha terpineol is not considered to show clastogenic potential.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance alpha terpineol which shares the same functional groups with the substance L-alpha terpineol also has comparable values for the relevant molecular properties.
See attached the reporting format - Reason / purpose for cross-reference:
- read-across source
- Target gene:
- Thymidine Kinase Gene
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 27-157% RTG
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 19-112% RTG
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Remarks on result:
- other: read-across from an analogue determined to be not mutagenic in mammalian cell assay
- Conclusions:
- Based on the read-across approach from the analogue alpha terpineol found negative in the Mouse Lymphoma Assay in the presense and in the absence of metabolic activation, laevo alpha terpienol can be considered not to be mutagenic in mammalian cells.
- Executive summary:
An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus (OECD test guideline 490) to test the potential of alpha terpineol to cause gene mutation. Treatment was performed for 4 hours with and without metabolic activation (±S9 mix). DMSO was used as solvent. The test item was examined previously in a cytotoxicity Test. Based on these results, the test item concentrations in the mutation assay ranged from 0.14 to 0.56 µL/mL. The experiments were performed using appropriate negative (vehicle) and positive control samples. Under conditions of the assay, the test substance was found negative in the presense and in the absence of metabolic activation. Based on these results, the read-across approach was applied and laevo alpha terpineol can be considered not to be mutagenic in mammalian cells.
Referenceopen allclose all
Table 1: Results of Test 1 (-S9Mix)
Metabolic activation |
Dose of test substance (μg/plate) |
Number of revertants (colony count / plate) |
|||||
Base pair substitution type |
Frameshift type |
||||||
TA 100 |
TA 1535 |
WP2uvrA |
TA 98 |
TA 1537 |
|||
S9 Mix (-) |
Negative control (DMSO) |
100 115 120 (112±10.4) |
12 13 9 (11±2.1) |
29 23 25 (26±3.1) |
19 13 13 (15±3.5) |
8 5 7 (7±1.5) |
|
39.1 |
90 99 97 (95±4.7 |
5 7 5 (6±1.2) |
26 31 38 (32±6.0) |
15 11 13 (13±2.0) |
4 5 6 (5±1.0) |
||
78.1 |
97 95 108 (100±7.0) |
7 12 10 (10±2.5) |
23 27 25 (25±2.0) |
13 12 13 (13±0.6) |
6 5 4 (5±1.0) |
||
156 |
100 103 99 (101±2.1) |
5 9 5 (6 ±2.3) |
36 29 42 (36±6.5) |
10 12 15 (12±2.5) |
8 5 11 (8±3.0) |
||
313 |
98 101 99 (99±1.5) |
5 6 11 (7±3.2) |
22 30 36 (29±7.0) |
13 10 11 (11±1.5) |
6 4 7 (6±1.5) |
||
625 |
46* 50* 47* (48±2.1) |
0* 0* 0* (0±0) |
27 21 27 (25±3.5) |
8* 8* 8* (8±0.0) |
0* 0* 0* (0±0) |
||
1250 |
0* 0* 0* (0±0) |
0* 0* 0* (0±0) |
0* 0* 0* (0±0) |
0* 0* 0* (0±0) |
0* 0* 0* (0±0) |
||
Positive controls |
S9Mix (without) |
Name |
AF-2 |
SAZ |
AF-2 |
AF-2 |
ICR-191 |
Dose (μg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
1.0 |
||
Number of colonies/ plate |
712 690 641 (681± 36.3) |
358 304 374 (345±36.7) |
115 123 117 (118±4.2) |
461 428 425 (438±20.0) |
1993 1905 1810 (1903±91.5) |
||
Notes:
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
SAZ: Sodium azide
ICR-191: 2-Methoxy-6-chloro-9-[ 3-(2-chloro ethyl)-aminopropylamino] acridine · 2HCL
*: Growth inhibition by the test substance was observed.
( ): The inside shows the average value and standard deviation of three plates
Table 2: Results of Test 1 (+S9Mix)
Metabolic activation |
Dose of test substance (μg/plate) |
Number of revertants (colony count / plate) |
|||||
Base pair substitution type |
Frameshift type |
||||||
TA 100 |
TA 1535 |
WP2uvrA |
TA 98 |
TA 1537 |
|||
S9 Mix (+) |
Negative control (DMSO) |
114 123 138 (125±12.1) |
8 9 7 (8±1.0) |
30 30 33 (31±1.7) |
22 27 26 (25±2.6) |
6 7 10 (8±2.1) |
|
9.77 |
NT |
11 10 7 (9±2.1) |
NT |
20 20 19 (20±0.6) |
8 7 10 (8±1.5) |
||
19.5 |
NT |
9 8 9 (9±0.6) |
NT |
18 21 26 (22±4.0) |
8 6 9 (8±1.5) |
||
39.1 |
116 121 110 (116±5.5) |
5 11 10 (9±3.2) |
22 31 38 (30±8.0) |
24 23 20 (22±2.1) |
5 8 8 (7±1.7) |
||
78.1 |
116 100 109 (108±8.0) |
11 10 8 (10±1.5) |
33 24 29 (29±4.5) |
21 20 24 (22±2.1) |
9 11 7 (9±2.0) |
||
156 |
110 91 111 (104±11.3) |
8 11 8 (9±1.7) |
26 25 23 (25±1.5) |
17 18 23 (19±3.2) |
7 6 8 (7±1.0) |
||
313 |
123 112 105 (113±9.1) |
5* 7* 7* (6±1.2) |
31 27 39 (32±6.1) |
22* 24* 18* (21±3.1) |
5* 7* 8* (7±1.5) |
||
625 |
90* 91* 103* (95±7.2) |
NT |
21 34 23 (26±7.0) |
NT |
NT |
||
1250 |
0* 0* 0* (0±0.0) |
NT |
0* 0* 0* (0±0.0) |
NT |
NT |
||
Positive controls |
S9Mix (with) |
Name |
B[α] P |
2AA |
2AA |
B[α] P |
B[α] P |
Dose (μg/plate) |
5.0 |
2.0 |
10.0 |
5.0 |
5.0 |
||
Number of colonies/ plate |
782 731 725 (746 ± 31. 3) |
359 288 344 (330±37.4) |
556 717 633 (635±80.8) |
338 337 376 (350±22.2) |
58 55 77 (63±11.9) |
||
Notes:
B[α] P: Benzo [α] pyrene
2AA: 2-Aminoanthracene
*: Growth inhibition by the test substance was observed.
NT: not tested
( ): The inside shows the average value and standard deviation of three plates.
Table 3: Results of Test 2 (-S9Mix)
Metabolic activation |
Dose of test substance (μg/plate) |
Number of revertants (colony count / plate) |
|||||
Base pair substitution type |
Frameshift type |
||||||
TA 100 |
TA 1535 |
WP2uvrA |
TA 98 |
TA 1537 |
|||
S9 Mix (-) |
Negative control (DMSO) |
103 104 116 (108±7.2) |
10 7 10 (9±1.7) |
38 38 41 (39±1.7) |
24 21 19 (21±2.5) |
7 10 8 (8±1.5) |
|
39.1 |
85 91 112 (96±14.2) |
10 13 12 (12±1.5) |
39 36 33 (36±3.0) |
22 20 20 (21±1.2) |
6 7 10 (8±2.1) |
||
78.1 |
92 88 95 (92±3.5) |
10 10 12 (11±1.2) |
39 49 38 (42±6.1) |
21 22 16 (20±3.2) |
8 8 8 (8±0.0) |
||
156 |
116 97 111 (108±9.8) |
7 10 7 (8±1.7) |
44 44 28 (39±9.2) |
18 21 20 (20±1.5) |
15 10 11 (12±2.6) |
||
313 |
104 117 112 (111±6.6) |
6 10 8 (8±2.0) |
41 38 47 (42±4.6) |
19 13 18 (17±3.2) |
10 10 9 (10±0.6) |
||
625 |
53* 44* 69* (55±12.7) |
2* 5* 3* (3±1.5) |
34 31 30 (32±2.1) |
10* 11* 10* (10±0.6) |
0* 0* 0* (0±0) |
||
1250 |
0* 0* 0* (0±0) |
0* 0* 0* (0±0) |
0* 0* 0* (0±0) |
0* 0* 0* (0±0) |
0* 0* 0* (0±0) |
||
Positive controls |
S9Mix (without) |
Name |
AF-2 |
SAZ |
AF-2 |
AF-2 |
ICR-191 |
Dose (μg/plate) |
0.01 |
0.5 |
0.01 |
0.1 |
1.0 |
||
Number of colonies/ plate |
637 692 650 (660± 28.7) |
293 309 353 (318±31.1) |
118 102 106 (109±8.3) |
439 428 474 (447±24.0) |
1548 1594 1489 (1544±52.6) |
||
Notes:
AF-2: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
SAZ: Sodium azide
ICR-191: 2-Methoxy-6-chloro-9-[ 3-(2-chloro ethyl)-aminopropylamino] acridine · 2HC1
*: Growth inhibition by the test substance was observed.
( ): The inside shows the average value and standard deviation of three plates.
Table 4: Results of Test 2 (+S9Mix)
Metabolic activation |
Dose of test substance (μg/plate) |
Number of revertants (colony count / plate) |
|||||
Base pair substitution type |
Frameshift type |
||||||
TA 100 |
TA 1535 |
WP2uvrA |
TA 98 |
TA 1537 |
|||
S9 Mix (+) |
Negative control (DMSO) |
120 108 114 (114±6.0) |
14 10 11 (12±2.1) |
42 44 30 (39±7.6) |
27 27 28 (27±0.6) |
13 10 10 (11±1.7) |
|
9.77 |
NT |
10 7 10 (9±1.7) |
NT |
28 25 22 (25±3.0) |
10 7 13 (10±3.0) |
||
19.5 |
NT |
7 10 6 (8±2.1) |
NT |
29 27 24 (27±2.5) |
9 7 8 (8±1.0) |
||
39.1 |
105 119 100 (108±9.8) |
11 11 10 (11±0.6) |
42 47 43 (44±2.6) |
25 30 27 (27±2.5) |
10 10 13 (11±1.7) |
||
78.1 |
125 114 116 (118±5.9) |
8 10 13 (10±2.5) |
51 40 39 (43±6.7) |
31 23 29 (28±4.2) |
7 6 7 (7±0.6) |
||
156 |
106 122 123 (117±9.5) |
8 9 6 (8±1.5) |
54 42 53 (50±6.7) |
30 29 29 (29±0.6) |
7 11 10 (9±2.1) |
||
313 |
119 133 114 (122±9.8) |
8* 10* 12* (10±2.0) |
39 37 41 (39±2.0) |
26* 27* 23* (25±2.1) |
8* 7* 7 (7±0.6) |
||
625 |
90* 104* 75* (90±14.5) |
NT |
38 38 47 (41±5.2) |
NT |
NT |
||
1250 |
0* 0* 0* (0±0.0) |
NT |
0* 0* 0* (0±0.0) |
NT |
NT |
||
Positive controls |
S9Mix (with) |
Name |
B[α] P |
2AA |
2AA |
B[α] P |
B[α] P |
Dose (μg/plate) |
5.0 |
2.0 |
10.0 |
5.0 |
5.0 |
||
Number of colonies/ plate |
727 771 804 (767 ± 38.6) |
379 339 352 (357±20.4) |
852 841 890 (861±25.7) |
288 266 253 (269±17.7) |
79 82 93 (85±7.4) |
||
Notes:
B[α] P: Benzo [α] pyrene
2AA: 2-Aminoanthracene
*: Growth inhibition by the test substance was observed.
NT: not tested
( ): The inside shows the average value and standard deviation of three plates.
Table 1. Testing of terpineol in the Salmonella/microsome assay
Dose (µg/plate) |
Number of revertants (Mean ± SD) |
|||||||
TA 100 |
TA 98 |
TA 97a |
TA 102 |
|||||
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
|
2500 |
- |
141±62* |
- |
4/0/0+ |
- |
- |
- |
- |
2000 |
53±14* |
146 ±2 |
- |
68± 13 |
- |
272 ±34 |
- |
833± 57* |
1500 |
165 ±16 |
212 ±28 |
43 ±5 |
60 ±10 |
225 ±32 |
248 ±12 |
- |
1573± 191 |
1250 |
158 ±19 |
188 ±23 |
42 ±2 |
58 ±2 |
224 ±11 |
246 ±6 |
862 ±187 |
1727± 108 |
1000 |
176 ± 6 |
208 ±33 |
40± 8 |
54± 10 |
191 ±8 |
254 ±21 |
1004 ±488 |
1429± 46 |
750 |
177 ±14 |
196 ±1 |
36 ±4 |
69 ±10 |
216 ±16 |
246 ±12 |
1418 ±176 |
1177± 42 |
500 |
177 ±12 |
161 ±24 |
28 ±7 |
53 ±10 |
195 ±13 |
241 ±16 |
1312± 89 |
1116± 74 |
250 |
186± 9 |
- |
30 ±6 |
- |
182 ±12 |
- |
812± 166 |
790± 47 |
100 |
- |
- |
- |
- |
- |
- |
924± 151 |
- |
50 |
- |
- |
- |
- |
- |
- |
737± 32 |
- |
25 |
- |
- |
- |
- |
- |
- |
611± 25 |
- |
0 |
176 ±14 |
197 ±7 |
40 ±7 |
70 ±6 |
158 ±2 |
214 ±18 |
731 ±36 |
771± 37 |
PC |
898 ± 51 |
742 ± 13 |
192 ± 31 |
312 ± 33 |
1098 ± 80 |
870 ± 140 |
4875 ± 1031 |
2024 ± 182 |
Dose O—Negative Control: 100 µl ethanol; PC—Positive Control: TA100/-S9, SA (0.5 µg/plate); TA100/+S9, 2AA (1 µg/plate); TA98/-S9, NPD (1 µg/plate); TA98/+S9; 2AA (0.5 µg/plate); TA97a/-S9, 4-NQNO (1 µg/plate); TA97a/+S9, 2AF (10 µg/plate); TA102/-S9, MC (0.5 µg/plate); TA102/+S9, B-alpha-P (50 µg/plate).
(-) Dose not tested.
(*) Toxicity apparent as an alteration of the background lawn.
(/+) mutant counts of individual plates.
Values are the means ±SD of three plates of one (out of two) representative experiment.
Table 2. Mutagenicity of terpineol to TA102 tester strain in the confirmatory experiment
Dose (µg/plate) |
- S9 |
+ S9 |
2000 |
- |
572 28* |
1500 |
|
1562 123 |
1250 |
- |
1526 219 |
1000 |
1050 38 |
1114 562 |
750 |
1088 32 |
1057 74 |
500 |
786 58 |
824 6 |
250 |
742 24 |
- |
100 |
634 26 |
- |
50 |
600 32 |
- |
25 |
630 54 |
- |
0 |
613 ±24 |
790± 120 |
PC |
5820 ± 311 |
1627± 260 |
Dose O—negative control (100 µl ethanol); PC—positive controls, -S9 (MC: 0.5 µg/plate); +S9 (B-alpha-P: 50 µg/plate)
(*) Toxicity apparent as an alteration of the background lawn.
Values are revertant counts (mean±SD of three plates).
Alpha terpineol was not mutagenic in all strains tested with and without metabolic activation.
Alpha terpineol was not mutagenic in all strains tested with and without metabolic activation.
Table 3 Salmonella Test Data
| Chemical Name | CAS # | Dose | TA98 | TA100 | TA1535 | TA1537 | TA1538 | |||||||||||
| no S9 | rat S9 | Ham'r S9 | no S9 | rat S9 | Ham'r S9 | no S9 | rat S9 | Ham'r S9 | no S9 | rat S9 | Ham'r S9 | no S9 | rat S9 | Ham'r S9 | ||||
| alpha-Terpineol | Negative | 98-55-5 | Negative | Negative | Negative | Negative | Negative | Negative | Negative | Negative | Negative | Negative | Negative | Negative | Negative | Negative | Negative | |
| DMSO | 13 ± 4 | 20 ± 3 | 23 ± 4 | 87 ± 5 | 97 ± 2 | 124 ± 7 | 19 ± 7 | 14 ± 4 | 14 ± 2 | 14 ± 4 | 6 ± 1 | 10 ± 1 | 10 ± 4 | 21 ± 6 | 14 ± 6 | |||
| 10ug | 20 ± 5 | 26 ± 3 | 24 ± 7 | 132 ± 7 | 118 ± 4 | 133 ± 9 | 24 ± 8 | 19 ± 4 | 22 ± 11 | 13 ± 1 | 11 ± 4 | 11 ± 2 | 20 ± 5 | 18 ± 2 | 23 ± 13 | |||
| 33ug | 25 ± 1 | 25 ± 4 | 29 ± 5 | 129 ± 14 | 106 ± 10 | 129 ± 4 | 22 ± 2 | 12 ± 5 | 22 ± 6 | 11 ± 1 | 13 ± 2 | 12 ± 3 | 15 ± 4 | 17 ± 1 | 25 ± 8 | |||
| 100ug | 17 ± 5 | 30 ± 5 | 28 ± 1 | 120 ± 11 | 113 ± 15 | 131 ± 15 | 29 ± 5 | 21 ± 4 | 22 ± 2 | 11 ± 1 | 12 ± 2 | 17 ± 3 | 16 ± 4 | 30 ± 1 | 28 ± 6 | |||
| 333ug | 16 ± 3 | 31 ± 1 | 31 ± 7 | 130 ± 10 | 117 ± 10 | 111 ± 21 | 30 ± 4 | 18 ± 5 | 6 ± 4 | 10 ± 6 | 19 ± 4 | 13 ± 3 | 17 ± 6 | 23 ± 4 | 24 ± 4 | |||
| 1000ug | 20 ± 3 | 31 ± 6 | 40 ± 4 | 111 ± 30 | 102 ± 20 | 127 ± 11 | 28 ± 3 | 15 ± 5 | 19 ± 4 | 9 ± 2 | 15 ± 2 | 13 ± 3 | 19 ± 7 | 21 ± 1 | 28 ± 1 | |||
| Positive 1 | 142 ± 18 | 435 ± 77 | 578 ± 43 | 451 ± 51 | 535 ± 48 | 576 ± 57 | 488 ± 24 | 173 ± 14 | 208 ± 19 | 223 ± 129 | 206 ± 58 | 403 ± 38 | 406 ± 37 | 754 ± 12 | 773 ± 118 | |||
| Positive 2 | 201 ± 13 | 953 ± 208 | 1516 ± 68 | 575 ± 67 | 1068 ± 121 | 1372 ± 77 | ||||||||||||
Table 1: Chromosome aberration in cultured Chinese hamster (CHL/IU) cells treated with Tepineol [Short-term treatment:+S9 mix]
Time (h) |
S9 mix |
Conc. Test article (µg/mL) |
Number of cells with structural chromosome aberration (%) |
Cell growth ratio (%) |
Number of cells with numerical chromosome growth aberration (%) |
|||||||||||||
Cells observed |
ctb |
cte |
csb |
cse |
other |
TA(%) |
g |
TAG(%) |
Judgement |
Cells observed |
Polyploid cells |
Other |
Total (%) |
Judgement |
||||
6-18 |
+ |
NC |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
100 |
100 |
0 |
0 |
0 |
- |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
109 |
100 |
0 |
0 |
0 |
|||||
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
(100) |
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|||||
100 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
2 |
2 |
- |
89 |
100 |
1 |
0 |
1 |
- |
||
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
79 |
100 |
1 |
0 |
1 |
|||||
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
2 (1.0) |
2 (1.0) |
(80) |
200 |
2 (1.0) |
0 (0.0) |
2 (1.0) |
|||||
200 |
100 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
1 |
- |
69 |
100 |
1 |
0 |
1 |
- |
||
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
69 |
100 |
0 |
0 |
0 |
|||||
200 |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
(66) |
200 |
1 (0.5) |
0 (0.0) |
1 (0.5) |
|||||
300 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
69 |
100 |
0 |
0 |
0 |
- |
||
100 |
1 |
0 |
0 |
0 |
0 |
1 |
1 |
2 |
69 |
100 |
0 |
0 |
0 |
|||||
200 |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
1 (0.5) |
2 (1.0) |
(66) |
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|||||
400 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
79 |
100 |
0 |
0 |
0 |
- |
||
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
69 |
100 |
0 |
0 |
0 |
|||||
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
(71) |
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|||||
500 |
100 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
1 |
- |
49 |
100 |
0 |
0 |
0 |
- |
||
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
49 |
100 |
0 |
0 |
0 |
|||||
200 |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
(47) |
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|||||
PC |
100 |
4 |
24 |
0 |
0 |
0 |
28 |
0 |
28 |
+ |
89 |
100 |
0 |
0 |
0 |
- |
||
100 |
3 |
22 |
0 |
0 |
0 |
25 |
0 |
25 |
99 |
100 |
0 |
0 |
0 |
|||||
200 |
7 (3.5) |
46 (23.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
53 (26.5) |
0 (0.0) |
53 (26.5) |
(90) |
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|||||
g: chromatid or chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange,
other: including fragmentation
TA: total number of cells with aberration excluding gap, TAG: total number of cells with aberration including gap.
NC: Negative control (DMSO)
PC: Positive control (cyclophosphamide, 14 μg/mL)
Each value in parenthesis on cell- growth ratio (%) data showed mean of cell-growth ratio against the negative control.
Table 2: Chromosome aberration in cultured Chinese hamster (CHL/IU) cells treated with Tepineol [Short-term treatment:-S9 mix]
Time (h) |
S9 mix |
Conc. Test article (µg/mL) |
Number of cells with structural chromosome aberration (%) |
Cell growth ratio (%) |
Number of cells with numerical chromosome growth aberration (%) |
|||||||||||||
Cells observed |
ctb |
cte |
csb |
cse |
other |
TA(%) |
g |
TAG(%) |
Judgement |
Cells observed |
Polyploid cells |
Other |
Total (%) |
Judgement |
||||
6-18 |
- |
NC |
100 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
1 |
- |
100 |
100 |
2 |
0 |
2 |
- |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
99 |
100 |
0 |
0 |
0 |
|||||
200 |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
(100) |
200 |
2 (1.0) |
0 (0.0) |
2 (1.0) |
|||||
100 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
74 |
100 |
0 |
0 |
0 |
- |
||
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
87 |
100 |
0 |
0 |
0 |
|||||
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
(81) |
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|||||
200 |
100 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
- |
49 |
100 |
0 |
0 |
0 |
- |
||
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
49 |
100 |
0 |
0 |
0 |
|||||
200 |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
(49) |
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|||||
300 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
49 |
100 |
0 |
0 |
0 |
- |
||
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
74 |
100 |
1 |
0 |
1 |
|||||
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
(62) |
200 |
1 (0.5) |
0 (0.0) |
1 (0.5) |
|||||
400 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
24 |
100 |
0 |
0 |
0 |
- |
||
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
24 |
100 |
0 |
0 |
0 |
|||||
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
(24) |
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|||||
PC |
100 |
3 |
28 |
0 |
0 |
0 |
31 |
0 |
31 |
+ |
112 |
100 |
0 |
0 |
0 |
- |
||
100 |
4 |
32 |
0 |
0 |
0 |
36 |
1 |
37 |
112 |
100 |
0 |
0 |
0 |
|||||
200 |
7 (3.5) |
60 (30.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
67 (33.5) |
1 (0.5) |
68 (34.0) |
(113) |
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|||||
g: chromatid or chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange,
other: including fragmentation
TA: total number of cells with aberration excluding gap, TAG: total number of cells with aberration including gap.
NC: Negative control (DMSO)
PC: Positive control (mitomycin C, 0.075 μg/mL)
Each value in parenthesis on cell- growth ratio (%) data showed mean of cell-growth ratio against the negative control.
Table 3: Chromosome aberration in cultured Chinese hamster (CHL/IU) cells treated with Tepineol [Continuous treatment: 24 h]
Time (h) |
S9 mix |
Conc. Test article (µg/mL) |
Number of cells with structural chromosome aberration (%) |
Cell growth ratio (%) |
Number of cells with numerical chromosome growth aberration (%) |
|||||||||||||
Cells observed |
ctb |
cte |
csb |
cse |
other |
TA(%) |
g |
TAG(%) |
Judgement |
Cells observed |
Polyploid cells |
Other |
Total (%) |
Judgement |
||||
24-0 |
- |
NC |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
100 |
100 |
1 |
0 |
1 |
- |
100 |
2 |
0 |
0 |
0 |
0 |
2 |
0 |
2 |
100 |
100 |
0 |
0 |
0 |
|||||
200 |
2 (1.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
2 (1.0) |
0 (0.0) |
2 (1.0) |
(100) |
200 |
1 (0.5) |
0 (0.0) |
1 (0.5) |
|||||
100 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
- |
100 |
100 |
1 |
0 |
1 |
- |
||
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
100 |
100 |
1 |
0 |
1 |
|||||
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
1 (0.5) |
(100) |
200 |
2 (1.0) |
0 (0.0) |
2 (1.0) |
|||||
200 |
100 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
- |
83 |
100 |
2 |
0 |
2 |
- |
||
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
83 |
100 |
1 |
0 |
1 |
|||||
200 |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
(83) |
200 |
3 (1.5) |
0 (0.0) |
3 (1.5) |
|||||
300 |
100 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
1 |
- |
49 |
100 |
1 |
0 |
1 |
- |
||
100 |
0 |
2 |
0 |
0 |
0 |
2 |
0 |
2 |
49 |
100 |
2 |
0 |
2 |
|||||
200 |
0 (0.0) |
3 (1.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
3 (1.5) |
0 (0.0) |
3 (1.5) |
(49) |
200 |
3 (1.5) |
0 (0.0) |
3 (1.5) |
|||||
400 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
33 |
100 |
1 |
0 |
1 |
- |
||
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
33 |
100 |
0 |
0 |
0 |
|||||
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
(33) |
200 |
1 (0.5) |
0 (0.0) |
1 (0.5) |
|||||
PC |
100 |
14 |
24 |
0 |
0 |
0 |
38 |
0 |
38 |
+ |
100 |
100 |
0 |
0 |
0 |
- |
||
100 |
11 |
24 |
0 |
0 |
0 |
35 |
0 |
35 |
116 |
100 |
0 |
0 |
0 |
|||||
200 |
25 (12.5) |
48 (24.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
73 (36.5) |
0 (0.0) |
73 (36.5) |
(108) |
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|||||
g: chromatid or chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange,
other: including fragmentation
TA: total number of cells with aberration excluding gap, TAG: total number of cells with aberration including gap.
NC: Negative control (DMSO)
PC: Positive control (mitomycin C, 0.050 μg/mL)
Each value in parenthesis on cell- growth ratio (%) data showed mean of cell-growth ratio against the negative control.
Table 4: Chromosome aberration in cultured Chinese hamster (CHL/IU) cells treated with Tepineol [Continuous treatment: 48 h]
Time (h) |
S9 mix |
Conc. Test article (µg/mL) |
Number of cells with structural chromosome aberration (%) |
Cell growth ratio (%) |
Number of cells with numerical chromosome growth aberration (%) |
|||||||||||||
Cells observed |
ctb |
cte |
csb |
cse |
other |
TA(%) |
g |
TAG(%) |
Judgement |
Cells observed |
Polyploid cells |
Other |
Total (%) |
Judgement |
||||
48-0 |
- |
NC |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
100 |
100 |
0 |
0 |
0 |
- |
100 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
1 |
99 |
100 |
0 |
0 |
0 |
|||||
200 |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
(100) |
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|||||
100 |
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
- |
92 |
100 |
1 |
0 |
1 |
- |
||
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
92 |
100 |
0 |
0 |
0 |
|||||
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
(92) |
200 |
1 (0.5) |
0 (0.0) |
1 (0.5) |
|||||
200 |
100 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
1 |
- |
76 |
100 |
0 |
0 |
0 |
- |
||
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
76 |
100 |
0 |
0 |
0 |
|||||
200 |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
(76) |
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|||||
300 |
100 |
1 |
0 |
0 |
0 |
0 |
1 |
0 |
1 |
- |
46 |
100 |
0 |
0 |
0 |
- |
||
100 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
53 |
100 |
1 |
0 |
1 |
|||||
200 |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
(50) |
200 |
1 (0.5) |
0 (0.0) |
1 (0.5) |
|||||
400 |
100 |
0 |
1 |
0 |
0 |
0 |
1 |
1 |
2 |
- |
23 |
100 |
0 |
0 |
0 |
- |
||
100 |
0 |
1 |
0 |
0 |
0 |
1 |
0 |
1 |
30 |
100 |
0 |
0 |
0 |
|||||
200 |
0 (0.0) |
2 (1.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
2 (1.0) |
1 (0.5) |
3 (1.5) |
(27) |
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|||||
PC |
100 |
13 |
56 |
0 |
0 |
0 |
69 |
0 |
69 |
+ |
107 |
100 |
3 |
0 |
3 |
- |
||
100 |
5 |
68 |
0 |
0 |
0 |
73 |
0 |
73 |
115 |
100 |
3 |
0 |
3 |
|||||
200 |
18 (9.0) |
124 (62.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
142 (71.0) |
0 (0.0) |
142 (71.0) |
(112) |
200 |
6 (3.0) |
0 (0.0) |
6 (3.0) |
|||||
g: chromatid or chromosome gap, ctb: chromatid break, cte: chromatid exchange, csb: chromosome break, cse: chromosome exchange,
other: including fragmentation
TA: total number of cells with aberration excluding gap, TAG: total number of cells with aberration including gap.
NC: Negative control (DMSO)
PC: Positive control (mitomycin C, 0.050 μg/mL)
Each value in parenthesis on cell- growth ratio (%) data showed mean of cell-growth ratio against the negative control.
Table 4 Mouse Lymphoma Test Data
| Non-Activated Cultures | S9-Activated Cultures | ||||||||||
| Chemical Name | Solvent | Dose (ug/mL) | Average TFT | Average VC | Mut Freq | RTG | Dose (ug/mL) | Average TFT | Average VC | Mut Freq | RTG |
| alpha-Terpineol | DMSO | 0.14 to 0.56 ul/mL | ul/mL | ||||||||
| 0,14 | 78 | 190 | 0,8 | 92 | 0,17 | 73 | 203 | 0,7 | 112 | ||
| 72 | 191 | 0,8 | 81 | 0,27 | 80 | 224 | 0,7 | 111 | |||
| 0,2 | 87 | 391 | 0,4 | 157 | 91 | 194 | 0,9 | 86 | |||
| 0,26 | 66 | 147 | 0,9 | 55 | 0,36 | 175 | 52 | ||||
| 74 | 192 | 0,8 | 75 | 86 | 238 | 0,7 | 89 | ||||
| 0,32 | 82 | 173 | 0,9 | 53 | 0,46 | 79 | 182 | 0,9 | 62 | ||
| 67 | 152 | 0,9 | 46 | 86 | 204 | 0,8 | 46 | ||||
| 0,38 | 71 | 161 | 0,9 | 27 | 0,56 | 68 | 247 | 0,6 | 19 | ||
| 71 | 180 | 0,8 | 29 | 196 | 23 | ||||||
| Solvent | 73 | 192 | 0,8 | Solvent | 62 | 193 | 0,6 | ||||
| Positive | 104 | 13 | 16,1 | 1 | Positive | 170 | 58 | 5,9 | 109 | ||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
In vitro gene mutation study in bacteria. Weight of Evidence: Read-across approach. Several Ames assays for mutagenecity on Salmonella typhimurium strains TA97a, TA 98, TA 100, TA 1535 TA 1537, TA 1538 and TA 102 were conducted on alpha terpineol and terpineol with the following results:
1) Terpineol was found to be not mutagenic in TA 100, TA 98 and TA 97a strains but weakly mutagenic in TA102 strain with and without metabolic activation.
2) Terpineol was found not mutagenic in TA100, TA98, 1535 and TA1537 and Escherichia coli strain WP2 uvrA with and without metabolic activation.
3) Alpha terpineol was found not mutagenic in TA 98, TA 100, TA 1535 and TA 1537 tester strains tested with and without metabolic activation.
4) Alpha terpineol was found not mutagenic in TA 1535, TA 1537, TA 1538, TA 98 and TA 100 tester strains tested with and without metabolic activation.
5) Alpha terpineol was found not mutagenic in TA1535, TA1537, TA1538, TA98 and TA100 tester strains tested with and without metabolic activation.
Based on these results, the read-across approach was applied and laevo alpha terpineol can be considered to be not mutagenic in bacteria with and without metabolic activation.
In vitro cytogenicity in mammalian cells.Weight of Evidence: Read-across approach. Terpineol was tested in an in vitro chromosome aberration study with cultured Chinese hamster lung CHL/IU cells in presence and absence of metabolic activation using a method according to OECD guideline 473. Based on a preliminary cytotoxicity study consisting on growth inhibition the tested concentrations were 500, 400, 300, 200, 100 and 0 (negative) μg/mL in the short-time treatment (+S9) and 400, 300, 200, 100 and 0 (negative) μg/mL in the short-time treatment (-S9) and continuous treatment (24 h and 48 h). Duplicate cultures were exposed to the test compound for 6 h (short time treatment) and for 24 h and 48 h (continuous treatment) before sampling. There were no statistically significant or dosage-related increases in both structural and numerical chromosome aberrations compared with the concurrent control (DMSO), with and without metabolic activation at both short-term and continuous treatment. Both positive controls, cyclophosphamide with metabolic activation and mitomycin C without metabolic activation, produced highly significant increases in the numbers of aberrant cells compared to control.From the above results, it was concluded that terpineol does not induce chromosome structural abnormality and chromosome number abnormality under the test conditions.Based on these results, the read-across approach was applied and laevo alpha terpineol is not considered to show clastogenic potential.
In vitro gene mutation study in mammalian cells. Weight of Evidence: Read-across approach. An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus (OECD test guideline 490) to test the potential of alpha terpineol to cause gene mutation. Treatment was performed for 4 hours with and without metabolic activation (±S9 mix). DMSO was used as solvent. The test item was examined previously in a cytotoxicity Test. Based on these results, the test item concentrations in the mutation assay ranged from 0.14 to 0.56 µL/mL. The experiments were performed using appropriate negative (vehicle) and positive control samples. Under conditions of the assay, the test substance was found negative in the presense and in the absence of metabolic activation. Based on these results, the read-across approach was applied and laevo alpha terpineol can be considered not to be mutagenic in mammalian cells.
Justification for classification or non-classification
Based on the available information, the substance is considered to be negative for genetic toxicity, and therefore the substance is not classified in accordance with CLP Regulation (EC) no 1272/2008.
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