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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames Test conducted according to OECD Guideline 471 and under GLP, the test item was not mutagenic up to cytotoxic concentrations in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E.coli WP2uvrA.

In an in vitro gene mutation study in mammalian cells conducted according to OECD Guideline 476 and under GLP, the test item was not mutagenic in the mouse lymphoma L5178Y test system.

In a chromosome aberration test conducted on human lymphocytes according to OECD Guideline 473 under GLP, the test item was not clastogenic up to cytotoxic concentrations.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 August 2014 - 06 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed according to OECD test guideline No. 471 and in compliance with GLP.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
OJ L 142/248
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
EPA 712-C-98-247
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK Department of Health (inspected on 01 - 03 July 2014 / signed on 16 September 2014)
Type of assay:
bacterial reverse mutation assay
Target gene:
S. typhimurium: Histidine gene
E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
No
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix (Test 1: 10% v/v and Test 2: 20% v/v)
Test concentrations with justification for top dose:
First Test: 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Second test: 50, 150, 500, 1500 and 5000 µg/plate

The highest dose, 5000 µg/plate, is the standard limit concentration recommended in the regulatory guidelines that this assay follows.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The solubility of Terpenic Oligomers was assessed at 50 mg/mL in water, dimethylsulphoxide (DMSO), acetone and ethanol. It was found to be insoluble in water and DMSO, but dissolved in acetone and ethanol. Acetone (analytical reagent grade) was used as the vehicle for this study.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9 mix, 2 µg/plate in DMSO for strains TA100 and TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix, 50 µg/plate in DMSO for strain TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without S9 mix, 2 µg/plate in DMSO for strain TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9 mix, 2 µg/plate in DMSO for strain WP2uvrA (pKM101)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9 mix, 5 µg/plate in DMSO for strains TA100 and TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9 mix, 10 µg/plate in DMSO for strain WP2uvrA (pKM101)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9 mix, 5 µg/plate in DMSO for strains TA98 and TA1537
Details on test system and experimental conditions:
TEST SYSTEM: The strains of S. typhimurium and E. coli were obtained from Moltox Inc. Batches of the strains were stored at ca -80°C as aliquots of nutrient broth cultures. Dimethylsulphoxide (DMSO) was added to the cultures at 8% v/v as a cryopreservative. Each batch of frozen strain was tested for amino acid requirement and, where applicable, for cell membrane permeability (rfa mutation), sensitivity to UV light, and the pKM101 plasmid, which confers resistance to ampicillin. The responses of the strains to a series of reference mutagens were also assessed.

METHOD OF APPLICATION: plate incorporation in agar (two independent tests)
- Cell density at seeding (if applicable): at least 10^9 per mL

DURATION :
- Preincubation period: 10 hours at 37°C with shaking
- Exposure duration: ca. 72 hours at approximately 37°C

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer).

ACCEPTANCE CRITERIA:
For a test to be considered valid:
- The mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range for the laboratory unless otherwise justified by the Study Director. The historical range is maintained as a rolling record over a maximum of five years.
- Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537, which have relatively low spontaneous reversion rates) that of the concurrent vehicle controls.
- Mean viable cell counts in the 10-hour bacterial cultures must be at least 10^9/mL.
Evaluation criteria:
- If exposure to a test substance produces a reproducible increase in revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) that of the concurrent vehicle controls, with some evidence of a positive concentration-response relationship, it is considered to exhibit mutagenic activity in this test system.
- If exposure to a test substance does not produce a reproducible increase in revertant colony numbers, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
- If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, even after additional testing, the test data may be subjected to analysis to determine the statistical significance of any increases in revertant colony numbers. The statistical procedures used are those described by Mahon et al (1989) and are usually Dunnett’s test followed, if appropriate, by trend analysis. Biological importance will be considered along with statistical significance. In general, treatment-associated increases in revertant colony numbers below two or three times those of the vehicle controls (as described above) are not considered biologically important. It should be noted that it is acceptable to conclude an equivocal response if no clear results can be obtained.
- Occasionally, these criteria may not be appropriate to the test data and, in such cases, the Study Director would use his/her scientific judgement.
Statistics:
None
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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, but tested up to precipitating concentrations
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 and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
HISTORICAL CONTROL DATA
- The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY
- Toxicity (observed as a reduction in revertant colony numbers) was seen in strain TA1537 following exposure to Terpenic Oligomers at 5000 μg/plate in the absence of S9 mix in the second test. No signs of toxicity towards any other tester strains were observed in either mutation test.
- Precipitate was observed on all plates containing Terpenic Oligomers at 5000 μg/plate in the presence of S9 mix in both tests and an oily residue was observed on all plates containing Terpenic Oligomers at 5000 μg/plate in the absence of S9 mix in both tests.

MUTAGENICITY RESULTS
No evidence of mutagenic activity was seen at any concentration of Terpenic Oligomers in either mutation test.
Remarks on result:
other: No evidence of mutagenic activity

None

Conclusions:
Terpenic Oligomers did not induce mutation in four histidine-requiring Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and one tryptophan-dependent Escherichia coli strain, WP2 uvrA (pKM101).
Executive summary:

In a reverse gene mutation assay performed according to OECD Guideline No. 471, EU Method B.13/14, US EPA Health Test Guideline OPPTS 870.5100 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA(pKM101) were treated with the test item using the Ames plate incorporation method. Up to seven dose levels were evaluated in triplicate with and without the addition of a rat liver homogenate metabolizing system (10% v/v liver S9 in standard co-factors) in a first test. As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The pre-incubation procedure is not suitable for acetone, which is toxic under such conditions. The variation used was, therefore, an increase in the S9 content of the S9 mix from 10% v/v to 20% v/v. The maximum concentration chosen was again 5000 μg/plate, but only five concentrations were used. These dose levels ranged from 50 to 5000 μg/plate.

Results of the first experiment did not show any evidence of toxicity following exposure to Terpenic Oligomers. Precipitate was observed on all plates containing Terpenic Oligomers at 5000 μg/plate in the presence of S9 mix and an oily residue was observed on all plates containing Terpenic Oligomers at 5000 μg/plate in the absence of S9 mix. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test.

No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Terpenic Oligomers at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

After the exposure period of the second experiment, toxicity (observed as a reduction in revertant colony numbers) was seen in strain TA1537 following exposure to Terpenic Oligomers at 5000 μg/plate in the absence of S9 mix. No signs of toxicity towards any other tester strains were observed. Precipitate was observed on all plates containing Terpenic Oligomers at 5000 μg/plate in the presence of S9 mix and an oily residue was observed on all plates containing Terpenic Oligomers at 5000 μg/plate in the absence of S9 mix.

No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Terpenic Oligomers at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

Therefore, Terpenic Oligomers did not induce mutation in four histidine-requiring Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and one tryptophan-dependent Escherichia coli strain, WP2 uvrA (pKM101), when tested at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines) in the absence and presence of a rat liver metabolic activation system (S-9).

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 August 2014 - 19 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
OJ L 142/262
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK Department of Health (inspected on 01 - 03 July 2014 / signed on 16 September 2014)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
thymidine kinase (tk) locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA)
- Rationale: Recommended test system in international guidelines (e.g. OECD, EC) and literature

MEDIA USED:
- Type of media:
R0: RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 µg/mL gentamicin.
R10p: R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v. R10p medium was used for cell culture unless otherwise specified.
R30p: R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.
R20p medium was used for the cloning efficiency plating. This was prepared by mixing equal volumes of R10p and R30p.
Selective medium consisted of R10p containing 4 µg/mL trifluorothymidine (TFT).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 2.4, 4.9, 9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625 and 1250 µg/mL
Without S9-mix, 24 hours treatment: 2.4, 4.9, 9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625 and 1250 µg/mL

Mutagenicitty test (3-hour exposure only):
Without S9-mix, 3 hours treatment: 9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625 and 1250 µg/mL
With S9-mix, 3 hours treatment: 9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625 and 1250 µg/mL
Without S9-mix, 24 hours treatment: 10, 30, 60, 80, 100, 125 and 150 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: A solubility test was performed. Terpenic Oligomers was found to be insoluble at 50 mg/mL in water and at 500 mg/mL in ethanol and dimethyl sulphoxide. Terpenic Oligomers was found to be soluble at 500 mg/mL in acetone. A solution of 500 mg/mL, dosed at 1% in medium, formed an oily residue. Terpenic Oligomers was dissolved and diluted in acetone (analytical reagent), shortly before dosing. The final volume of acetone added to the cultures was 1% (v/v).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9-mix: 10 µg/mL (3-hour exposure) and 5 µg/mL (24-hour exposure) in dimethyl sulfoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
With S9-mix: 1 µg/mL (3-hour exposure) in dimethyl sulfoxide
Details on test system and experimental conditions:
CLEANSING
Spontaneous thymidine kinase deficient mutants, TK -/-, were eliminated from the cultures by a 24-hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196°C, in heat-inactivated donor horse serum (HiDHS) containing 10% DMSO.

DOSE RANGE FINDING TEST
Cells were exposed to the test substance for 3 hours in the absence and presence of S9 mix and for 24 hours in the absence of S9 mix.
For 3-hour exposures, cultures contained a total of 6 x 10^6 cells. The final volume of the cultures was 5 mL and the final concentration of the S9 fraction was 2% v/v, if present. For 24-hour exposures, cultures contained a total of 1.5 x 10^6 cells in a total volume of 5 mL. One culture was prepared for each concentration of the test substance for each test condition. Vehicle controls were tested in duplicate for each test condition.
The test substance was formulated and serially diluted in the solvent. Aliquots of 50 µL of test substance dilution (at 100 times the desired final concentration) or vehicle were added to each culture prior to incubation for 3 hours (continuous shaking at 37°C) or 24 hours (static incubator, at 37°C, 5% (v/v) CO2). At the end of the 3-hour exposure period, the cells were washed once, resuspended in R10p to nominally 2 x 10^5 cells/mL (assuming no cell loss), incubated (at 37°C, 5% (v/v) CO2) and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary. At the end of the 24-hour exposure period, the cells were washed once, resuspended in 5 mL R10p and counted, to ascertain treatment growth. The cultures were then diluted to 2 x 10^5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary.
The RSG was used to determine the concentrations of test substance used in the main test; ideally the maximum concentration should reduce RTG to approximately 10 to 20% of the concurrent vehicle control value. There was no evidence of toxicity in the preliminary toxicity test following a 3-hour exposure, so the maximum concentration tested in the absence and presence of S9 mix was 1250 µg/mL. There was evidence of toxicity in the preliminary toxicity test following a 24-hour exposure, so the maximum concentrations tested in the 24-hour exposure in the absence of S9 mix was 150 µg/mL. The formulations were added at 1% final volume in medium.

MUTAGENICITY TEST: 3-hour exposure in the absence and presence of S9 mix
The procedure for preparing the cell suspension was the same as for the preliminary toxicity test. Cultures contained a total of 1.2 x 10^7 cells in a final volume of 10 mL. The final concentration of the S9 fraction was 2% v/v, if present. Duplicate cultures were prepared throughout for each concentration of test substance and positive control. Quadruplicate cultures were prepared for vehicle controls. Aliquots of 100 µL of test substance dilution (at 100 times the desired final concentration), vehicle or positive control were added, then all cultures were incubated, with continuous shaking, for 3 hours at 37°C. At least four serial dilutions of the test substance were tested.

Following the 3-hour exposure, the cells were washed once, resuspended in R10p to nominally 2 x 10^5 cells/mL (assuming no cell loss) and incubated for a further 48 hours to allow for expression of mutant phenotype. The cultures were sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p where necessary. After 48 hours cultures with a density of more than 1 x 10^5 cells/mL were assessed for cloning efficiency (viability) and mutant potential by plating in 96-well plates. Cultures that were assessed were chosen at the Study Director’s discretion. Cloning efficiency was assessed by plating 1.6 cells/well in R20p, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 10^3 cells/well in selective medium, two plates being prepared per culture. The plates were placed in a humidified incubator at 37°C in an atmosphere of 5% CO2 in air.

After the plates had been incubated for at least 7 days for viability plates and approximately 10 to 14 days for mutant plates, the number of empty wells was assessed for each 96-well plate (P0). P0 was used to calculate the cloning efficiency (CE) and mutant frequency (MF). The colony size distribution in the vehicle and positive controls was examined to ensure that there was an adequate recovery of small colony mutants. The maximum concentration assessed for mutant frequency in the main test was 1250 µg/mL in the absence and presence of S9 mix.

MUTAGENICITY TEST: 24-hour exposure in the absence of S9 mix
A test with a 24-hour exposure in the absence of S9 mix was carried out. Duplicate 10 mL cultures containing 3 x 10^6 cells were treated for 24 hours with 100 µL of test substance or positive control. Quadruplicate cultures were prepared for vehicle controls. At the end of the exposure period, the cells were washed once, resuspended in 10 mL R10p and counted to ascertain treatment growth. The cultures were then diluted to 2 x 10^5 cells/mL with R10p as appropriate, incubated and sampled after 24 and 48 hours to assess growth in suspension. After sampling at 24 hours the cell density was readjusted to 2 x 10^5 cells/mL with R10p, the intention being to retain at least 1 x 10^7 cells. Following this, the procedure was the same as in the 3-hour treatment. The maximum concentration assessed for mutant frequency in the main test was 100 µg/mL.

NUMBER OF REPLICATIONS: Duplicates for test samples and positive controls. Four replicates for the vehicle control.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER:
Prior to commencing testing, the osmolality of the test substance in medium was tested at concentrations of 1250, 2500 and 5000 µg/mL. The pH of the test substance in medium was also tested at concentrations of 1250 µg/mL.
Evaluation criteria:
The following criteria were applied for assessment of individual assay results using data for MF where the RTG normally exceeded 10%:

Definitions:
GEF = Global Evaluation Factor. For microwell assays this is 126 x 10^-6 (Moore et al., 2006).
The assay was considered valid in accordance with the assay acceptance criteria.

- The test agent was regarded as negative if:
*The mean mutant frequency of all test concentrations was less than the sum of the mean concurrent vehicle control mutant frequency and the GEF.

- If the mutant frequency of any test concentrations exceeded the sum of the mean concurrent solvent control mutant frequency and the GEF, a linear trend test was applied:
*If the linear trend test was negative, the result was regarded as negative.
*If the linear trend test was positive, this indicated a positive, biologically relevant response.

Where appropriate, other factors were considered in the interpretation of the results, for example, the reproducibility within and between tests, the overall number of mutant colonies (as opposed to mutation frequency) and the nature of any concentration-related effect(s).

Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis. In cases where the results were inconclusive, further testing and/or a test modification may have been required to better define the assay response.
Statistics:
The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989).
Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend (p<0.05).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
3-hour exposure with and without S9 mix
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:
mouse lymphoma L5178Y cells
Remarks:
24-hour exposure
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The upper two dose levels of 125 and 150 μg/ml were not plated for mutation frequency because of excessive toxicity.
Vehicle controls validity:
valid
Untreated negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No fluctuations in pH of the medium of more than 1 unit were observed compared with the vehicle control. The maximum final concentration tested in the preliminary toxicity test was 1250 µg/mL.
- Effects of osmolality: No fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control at 1250 µg/mL; 2500 and 5000 µg/mL caused a change of greater than 50 mOsm/kg.
- Precipitation:
*Preliminary toxitiy test: Precipitate was observed at concentrations of 156.3 µg/mL and greater in the absence and presence of S9 mix, following a 3-hour exposure. Following a continuous exposure for 24 hours, precipitation (assessed by eye at the end of treatment) was observed at concentrations of 156.3 µg/mL and greater.
*Main mutation test - 3-hour treatment in absence and in presence of S9 mix: Precipitation was observed at concentrations of 156.3 µg/mL and greater.
*Main mutation test - 24-hour treatment in absence of S9 mix: No precipitate was observed by eye at the end of treatment.

PRELIMINARY TOXICITY TEST:
A final concentration of 1250 µg/mL, dosed at 1%v/v, was used as the maximum concentration in the preliminary toxicity test, in order to test up to the limit concentration within this test system due to a change in osmolality observed at higher concentrations.
Exposure to Terpenic Oligomers at concentrations from 2.4 to 1250 µg/mL in the absence and presence of S9 mix (3-hour exposure) resulted in no reduction in relative suspension growth (RSG) values. Exposure to concentrations from 2.4 to 1250 µg/mL (24-hour exposure) resulted in RSG values from 106 to 1%.
Concentrations used in the main test were based upon these data.

MAIN MUTATION TEST: 3-hour treatment in the absence of S9 mix
Cultures were exposed to Terpenic Oligomers at concentrations from 9.8 to 1250 µg/mL and were assessed for determination of mutation frequency. The cultures assessed contained 3 concentrations that did not contain precipitate and 2 concentrations that contained precipitate. A judgement was also made on the heterogeneity of the observed suspension growth.
No reductions in relative total growth (RTG) values were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity.

MAIN MUTATION TEST: 3-hour treatment in the presence of S9 mix
Cultures were exposed to Terpenic Oligomers at concentrations from 9.8 to 1250 µg/mL and were assessed for determination of mutation frequency. The cultures assessed contained 3 concentrations that did not contain precipitate and 2 concentrations that contained precipitate. A judgement was also made on the heterogeneity of the observed suspension growth. No reductions in relative total growth (RTG) values were obtained relative to the vehicle control.
There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.
The results obtained in response to the exposure of cultures to Terpenic Oligomers in the presence of S9 mix did not demonstrate mutagenic potential. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. All mean mutant frequencies of the test concentrations were within the historical vehicle control values and there were no clear increases in the mean mutant frequencies of any test concentration assessed that were associated with a linear trend (P>0.05). Therefore it was considered not to be beneficial to perform a direct repeat of the assay.

MAIN MUTATION TEST: 24-hour treatment in the absence of S9 mix
Cultures were exposed to Terpenic Oligomers at concentrations from 10 to 150 µg/mL and were assessed for determination of mutation frequency. Higher concentrations of 125 and 150 μg/mL were not continued due to their excessive toxicity shown suspension growth. RTG values from 135 to 18% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA:
The positive controls, methyl methanesulphonate and benzo[a]pyrene, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants. In all tests the concurrent vehicle and positive control were within acceptable ranges.
Remarks on result:
other: No evidence of mutagenic potential

None

Conclusions:
Terpenic oligomers did not demonstrate mutagenic potential in this in vitro cell mutation assay.
Executive summary:

The mutagenic activity of Terpenic oligomers was investigated in an in vitro mammalian cell gene mutation test conducted with L5178Y mouse lymphoma cells in accordance with OECD Guideline No. 490, EU Method B.17 and EPA OPPTS 870.5300 and under GLP compliance. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).

The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

The test item was found to be soluble at 500 mg/mL in acetone. A final concentration of 1250 µg/mL, dosed at 1%v/v, was used as the maximum concentration in the preliminary toxicity test, in order to test up to the limit concentration within this test system due to a change in osmolality observed at higher concentrations.

Toxicity was observed in the preliminary toxicity test. Precipitate (observed by eye at the end of exposure) of the test substance was observed at and above 156.3 μg/ml in all three exposure groups. Following a 3-hour exposure to Terpenic oligomers at concentrations from 2.4 to 1250 µg/mL, no reduction in relative suspension growth (RSG) was observed in the absence and presence of S9 mix respectively. Following a 24-hour exposure in the absence of S9 mix RSG was reduced from 106 to 1%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG) (24-hour exposure), or to test up the standard limit concentration within this test system as recommended in the regulatory guidelines (3-hour exposures).

Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentration assessed for mutant frequency in the 3-hour treatment in the absence and presence of S9 mix was 1250 µg/mL, corresponding to the limit concentration within this test system due to a change in osmolality observed at higher concentrations. In the absence and presence of S9 mix a reduction in RTG was not observed.

In the 24-hour treatment, the maximum concentration assessed for mutant frequency was 100 µg/mL, the maximum dose level was limited by toxicity. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. The RTG was reduced to 18%.

In all tests the concurrent vehicle and positive control were within acceptable ranges.

Therefore, Terpenic oligomers did not demonstrate mutagenic potential in this in vitro cell mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 August 2014 - 19 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 473 without any deviation.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
OJ L 142/225
Deviations:
no
Qualifier:
according to guideline
Guideline:
other:
Version / remarks:
OECD Guideline for the Testing of Chemicals. (Dec 2013) Proposal for updating test guideline 473: In Vitro Mammalian Chromosome Aberration Test
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK Department of Health (inspected on 01 - 03 July 2014 / signed on 16 September 2014)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: human
Remarks:
primary culture
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Colcemid®
Metabolic activation:
with and without
Metabolic activation system:
5 % S9 (final concentration); S9 fraction was obtained from the liver homogenates of male Sprague-Dawley rats treated with phenobarbitone and 5,6-benzoflavone
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
0,12.6, 21, 34.99, 58.32, 97.2, 162, 270, 450, 750 and 1250 μg/mL; 3h treatment period with and without metabolic activation, and a continuous exposure of 21 h without metabolic activation

Justification for the highest analysed concentration was based on the highest final concentration for the test system, where no change of greater than 50 mOsmol/kg when compared to the concurrent vehicle control. Otherwise, selection was determined by cytotoxicity.

Main Test:
Without S9, 3-hour treatment followed by 18-hour incubation in fresh medium: 0, 250, 500, 750, 1000 and 1250 μg/mL
With S9 (5%), 3-hour treatment followed by 18-hour incubation in fresh medium: 0, 250, 500, 750, 1000 and 1250 μg/mL
Without S9, 21-hour treatment: 0, 15.63, 31.25, 62.5, 125, 250, 500, 750, 1000 and 1250 μg/mL

From these results the concentration causing a decrease in mitotic index of at least 50% (where possible) of the vehicle control value was the highest concentration selected for metaphase analysis. Intermediate and low concentrations were also selected.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: Terpenic Oligomers was found to be soluble at 500 mg/mL in acetone. Acetone (analytical reagent) was, therefore, used as the vehicle for this study.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9 mix, 0.2 µg/mL (3-hour treatment) and 0.1 µg/mL (21-hour treatment) in sterile purified water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9 mix, 7.5 µg/mL (3-hour treatment) in sterile purified water
Details on test system and experimental conditions:
TEST SYSTEM:
Human blood was collected aseptically from two healthy, non-smoking, adult donors, pooled (in equal volumes from each donor) and diluted with RPMI 1640 tissue culture medium supplemented with 10% foetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM L-glutamine. As lymphocytes do not normally undergo cell division, they were stimulated to do so by the addition of phytohaemagglutinin (PHA), a naturally occurring mitogen.

CELL CULTURE:
Cultures were established from the prepared (pooled) sample and dispensed as 5 mL aliquots (in sterile universal containers) so that each contained blood (0.4 mL), culture medium (4.5 mL) and PHA solution (0.1 mL). All cultures were then incubated at 37°C, and the cells resuspended (at least once daily) by gentle inversion.

DURATION
- Preincubation period: ca. 48 hours
- Exposure duration: 3h with or without S9 mix and 21h without S9 mix (Preliminary test and Main test)
- Fixation time (start of exposure up to fixation or harvest of cells): ca. 2 hours

SPINDLE INHIBITOR (cytogenetic assays): Mitotic activity was arrested by addition of demecolcine (Colcemid 0.1 μg/mL), two hours before the harvest time.

STAIN (for cytogenetic assays): 10 % Giemsa prepared in buffered water (pH 6.8).

NUMBER OF REPLICATIONS:
Preliminary toxicity test: Duplicate cultures were used for treatment with the vehicle, and single cultures for treatment with the test substance for each test condition. No positive control cultures were prepared.
Main experiments: Duplicate cultures/dose

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The pellets were resuspended, then centrifuged at 500g for 5 minutes and finally resuspended in a small volume of fresh fixative. A few drops of the cell suspensions were dropped onto pre-cleaned microscope slides and allowed to air dry. The slides were then stained in 10%
Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and mounted in DPX. The remainder of the cell pellets in fixative were stored at approximately 4°C until slide analysis was completed.

NUMBER OF CELLS EVALUATED:
The prepared slides were examined by light microscopy using a low power objective. The proportion of mitotic cells per 1000 cells in each culture was recorded (except for positive control treated cultures, or when clear evidence of overt toxicity was observed, or in cultures where there were no signs of cytotoxicity).

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):
One hundred and fifty metaphase figures were examined from each culture, however, this number was reduced in cultures showing a high level of aberrant cells, where 15 cells in 150 metaphases with structural aberrations (excluding gaps) were observed. Chromosome aberrations were scored according to the classification of the ISCN (2009). Only cells with 44 - 48 chromosomes were analysed. The vernier readings of all aberrant metaphase figures were recorded.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: If greater than 44 chromosomes are scored and the number is a multiple of the haploid count then the cell is classified as a polyploid cell.
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as endoreduplicated (E).
- The incidence of polyploid and endoreduplicated cells (i.e. the ploidy status) was recorded as a percentage of the 150 metaphases analysed per slide.

OTHER: An additional test was performed in the presence of S9 mix (3-hour treatment) due to a low level of aberrant cells in response to treatment with Cyclophosphamide.
Evaluation criteria:
An assay is considered to be acceptable if the negative and positive control values lie within the current historical control range.

The test substance is considered to cause a positive response if the following conditions are met:
- Statistically significant increases (p<0.01) in the frequency of metaphases with aberrant chromosomes (excluding gaps) are observed at one or more test concentration.
- The increases exceed the vehicle control range of this laboratory, taken at the 99% confidence limit.
- The increases are reproducible between replicate cultures.
- The increases are not associated with large changes in pH, osmolality of the treatment medium or extreme toxicity.
- Evidence of a concentration-related response is considered to support the conclusion.

A negative response is claimed if no statistically significant increases in the number of aberrant cells above concurrent control frequencies are observed, at any concentration.

A further evaluation may be carried out if the above criteria for a positive or a negative response are not met.
Statistics:
- The number of aberrant metaphase cells in each test substance group was compared with the vehicle control value using the one-tailed Fisher exact test (Fisher 1973).

- A Cochran-Armitage test for trend (Armitage, 1955) was applied to the control and all test substance groups. If this is significant at the 1% level, the test is reiterated excluding the highest concentration group - this process continues until the trend test is no longer significant.

- D20’s (the minimum concentration (mg/mL) at which aberrations were found in 20% of metaphases) were estimated (where possible) using logistic regression on a log(concentration) scale, allowing the number of control aberrations to be non-zero (Armitage et al., 2002). The following model was used:
p = C + (1 – C) / (1 + exp[– intercept – slope ln(conc)])
p is the proportion of cells with aberrations, conc is the concentration of test substance. C is a parameter estimating the control proportion of aberrations.
As Terpenic Oligomers did not demonstrate any evidence of causing an increase in the frequency of structural chromosome aberrations under any treatment condition, no D20 values for structural aberrations are presented.

- The data was analysed using the SAFEStat (SAS statistical applications for end users, version 1.1) Chromosome Aberrations application (version 1.1) which was developed in SAS (SAS INSTITUTE 2002).
Key result
Species / strain:
lymphocytes: Human
Remarks:
Primary culture
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: Human
Remarks:
Primary culture
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Reduction in the mitotic index to 54% of the vehicle control value at 500 μg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No fluctuations in pH of the medium of more than 1 unit were observed compared with the vehicle control.
- Effects of osmolality: No fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control at 1250 µg/mL; 2500 and 5000 µg/mL caused a change of greater than 50 mOsm/kg.
- Precipitation: No

PRELIMINARY TOXICITY TEST:
In the absence of S9 mix following 3-hour treatment, Terpenic Oligomers caused no biologically significant reduction in the mitotic index at 1250 μg/mL, the highest tested concentration (due to a change of greater than 50 mOsmol/kg at higher concentrations), when compared to the vehicle control.
In the presence of S9 mix following 3-hour treatment, Terpenic Oligomers caused a reduction in the mitotic index to 74% of the vehicle control value at 1250 μg/mL. the highest tested
concentration (due to a change of greater than 50 mOsmol/kg at higher concentrations).
In the absence of S9 mix following 21-hour continuous treatment, Terpenic Oligomers caused a reduction in the mitotic index to 52% of the vehicle control value at 1250 μg/mL.
The concentrations used in the main test were based upon these data.

COMPARISON WITH HISTORICAL CONTROL DATA: All positive and vehicle solvent controls were in the range of historical laboratory control data.

MAIN STUDY RESULTS:
Main test: In the absence of S9 mix, 3-hour treatment
- Terpenic Oligomers caused no significant reduction in the mitotic index at 1250 μg/mL, compared to the vehicle control value. The concentrations selected for the metaphase analysis were 750, 1000 and 1250 μg/mL.
- Terpenic Oligomers caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any analysed concentration, when compared to the vehicle
control.
- No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared to the vehicle control.

Main test: In the presence of S9 mix, 3-hour treatment
- Terpenic Oligomers caused a reduction in the mitotic index to 76% of the vehicle control value at 1250 μg/mL. The concentrations selected for metaphase analysis were 250, 1000 and 1250 μg/mL.
- Terpenic Oligomers caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any analysed concentration, when compared to the vehicle
control.
- No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared to the vehicle control.

Additional Main test: In the presence of S9 mix, 3-hour treatment
- Terpenic Oligomers caused a reduction in the mitotic index to 64% of the vehicle control value at 1250 μg/mL. The concentrations selected for metaphase analysis were 750, 1000 and 1250 μg/mL.
- Terpenic Oligomers caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any analysed concentration, when compared to the vehicle
control.
- No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared to the vehicle control.

Main test: In the absence of S9 mix, 21-hour continuous treatment
- Terpenic Oligomers caused a reduction in the mitotic index to 54% of the vehicle control value at 500 μg/mL. The concentrations selected for metaphase analysis were 31.25, 250 and 500 μg/mL.
- Terpenic Oligomers caused no statistically significant increases in the proportion of cells with chromosomal aberrations at any analysed concentration, when compared to the vehicle control.
- No statistically significant increases in polyploid or endoreduplicated metaphases were observed during metaphase analysis, when compared to the vehicle control.
Remarks on result:
other: 3-hour exposure
Remarks:
no evidence of causing an increase in the frequency of structural chromosome aberrations

7.6.1c/1. Summary of results of the main test

 Exposure period (hours)     S9 mix   

 Nominal concentration of

Terpebnic Oligomers (µg/mL)   

 Cells with aberrations excluding gaps        Cells with aberrations including gaps        Relative Mitotic Index (%)   
 Individual values (%)     Mean (%)  Individual values (%)     Mean (%)
 3

 0 (Acetone)

750

1000

1250

0.2 (Mitomycin C)

 0.0

0.0

0.0

0.7

14.2

0.0

0.7

0.7

1.3

12.2 

 0.0

0.3

0.3

1.0

13.1***

0.0

0.0

0.0

1.3

14.2 

0.7

0.7

0.7

2.0

12.2 

0.3

0.3

0.3

1.7

13.1*** 

100

114

113

102

95 

 3  +

 0 (Acetone)

250

1000

1250

7.5 (Cyclophosphamide)

0.7

0.0

0.7

0.0

6.7  

0.0

0.7

0.0

0.0

8.0 

0.3

0.3

0.3

0.0

7.3*** 

0.7

0.0

0.7

0.0

7.3 

0.0

1.3

0.0

0.0

9.3 

 0.3

0.7

0.3

0.0

8.3***

100

107

84

76

82 

 21  -

 0 (Acetone)

31.25

250

500

0.1 (Mitomycin C)

 0.7

0.0

0.0

0.0

18.8

0.7

0.0

0.0

0.0

21.4 

0.7

0.0

0.0

0.0

20.0*** 

1.3

0.0

0.0

0.7

18.8 

0.7

0.0

0.0

0.7

21.4 

1.0

0.0

0.0

0.7

20.0*** 

 100

113

75

54

99

One-tailed Fisher's exact test

*** p<0.001

Otherwise p>=0.05

7.6.1c/2. Summary of results of the main test (additional test)

 Exposure period (hours)     S9 mix     Nominal concentration of Terpenic Oligomers (µg/mL)     Cells with aberrations excluding gaps        Cells with aberrations including gaps        Relative Mitotic Index (%)   
  Individual values (%)    Mean (%)  Individual values (%)     Mean (%)
 3  +

 0 (Acetone)

750

1000

1250

7.5 (Cyclophosphamide)

0.7

0.0

1.3

0.0

27.8 

0.0

0.0

0.0

0.0

21.7 

0.3

0.0

0.7

0.0

24.4*** 

0.7

0.0

1.3

0.7

31.5 

0.7

0.7

0.7

0.0

23.2 

 0.7

0.3

1.0

0.3

26.8***

100

101

93

64

83 

One-tailed Fisher's exact test

*** p<0.001

Otherwise p>=0.05

Conclusions:
Test item Terpenic oligomers has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured human lymphocytes were exposed to the test item at the concentrations below.

Preliminary Toxicity Test (in order to determine the toxicity of Terpenic oligomers to cultured human lymphocytes):

0,12.6, 21, 34.99, 58.32, 97.2, 162, 270, 450, 750 and 1250 μg/mL; 3h treatment period with and without metabolic activation, and a continuous exposure of 21 h without metabolic activation

Main Test

Without S9, 3-hour treatment followed by 18-hour incubation in fresh medium: 0, 250, 500, 750, 1000 and 1250 μg/mL

With S9 (5%), 3-hour treatment followed by 18-hour incubation in fresh medium: 0, 250, 500, 750, 1000 and 1250 μg/mL

Without S9, 21-hour treatment: 0, 15.63, 31.25, 62.5, 125, 250, 500, 750, 1000 and 1250 μg/mL

Mitotic index data was used to estimate the test item toxicity and for selection of the dose levels for the main test.

Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared to examine mitotic indices and chromosomal aberrations. Vehicle and positive controls were also included in this test.

All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. Both positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

In the absence and presence of S9 mix following 3-hour treatment, Terpenic oligomers caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any analysed concentration, when compared to the vehicle control.

In the absence of S9 mix following 21-hour continuous treatment, Terpenic oligomers caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any analysed concentration, when compared to the vehicle control.

No statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis, under any treatment condition, when compared to the vehicle control.

Therefore, Test item Terpenic oligomers has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Three key studies were identified for the genetic toxicity assesment (two for mutagenicity potential and one for clastogenicity potential).

Gene mutation assay:

A reverse gene mutation assay performed according to OECD Guideline No. 471, EU Method B.13/14, US EPA Health Test Guideline OPPTS 870.5100 and in compliance with GLP. Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA (pKM101) were treated with the test item using the Ames plate incorporation method. Up to seven dose levels (five dose levels for the second test) were evaluated in triplicate with and without the addition of a rat liver homogenate metabolizing system into two individual tests.

Results of the first experiment did not show any evidence of toxicity following exposure to Terpenic oligomers. Precipitate was observed on all plates containing Terpenic oligomers at 5000 μg/plate in the presence of S9 mix and an oily residue was observed on all plates containing Terpenic oligomers at 5000 μg/plate in the absence of S9 mix. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test.

No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Terpenic oligomers at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

After the exposure period of the second experiment, toxicity (observed as a reduction in revertant colony numbers) was seen in strain TA1537 following exposure to Terpenic oligomers at 5000 μg/plate in the absence of S9 mix. No signs of toxicity towards any other tester strains were observed. Precipitate was observed on all plates containing Terpenic oligomers at 5000 μg/plate in the presence of S9 mix and an oily residue was observed on all plates containing Terpenic Oligomers at 5000 μg/plate in the absence of S9 mix.

No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to Terpenic oligomers at any concentration up to and including 5000 μg/plate in either the presence or absence of S9 mix.

The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

Therefore, test item Terpenic oligomers did not induce mutation in four histidine-requiring Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and one tryptophan-dependent Escherichia coli strain, WP2 uvrA (pKM101), when tested under the conditions of this study including treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines) in the absence and presence of a rat liver metabolic activation system (S-9).

The mutagenic activity of Terpenic oligomers was also investigated in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells in accordance with OECD Guideline No. 490, EU Method B.17 and EPA OPPTS 870.5300 and under GLP compliance. The study consisted of a preliminary toxicity test and three independent mutagenicity assays. The cells were exposed for either 3 hours or 24 hours in the absence of exogenous metabolic activation (S9 mix) or 3 hours in the presence of S9 mix.

Toxicity was observed in the preliminary toxicity test. Precipitate (observed by eye at the end of exposure) of the test substance was observed at and above 156.3 μg/ml in all three exposure groups. Following a 3-hour exposure to Terpenic oligomers at concentrations from 2.4 to 1250 µg/mL, no reduction in relative suspension growth (RSG) was observed in the absence and presence of S9 mix respectively. Following a 24-hour exposure in the absence of S9 mix RSG was reduced from 106 to 1%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG) (24-hour exposure), or to test up the standard limit concentration within this test system as recommended in the regulatory guidelines (3-hour exposures).

Following 3-hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentration assessed for mutant frequency in the 3-hour treatment in the absence and presence of S9 mix was 1250 µg/mL, corresponding to the limit concentration within this test system due to a change in osmolality observed at higher concentrations. In the absence and presence of S9 mix a reduction in RTG was not observed.

In the 24-hour treatment, the maximum concentration assessed for mutant frequency was 100 µg/mL, the maximum dose level was limited by toxicity. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF. The RTG was reduced to 18%.

In all tests the concurrent vehicle and positive control were within acceptable ranges.

Therefore, Terpenic oligomers did not demonstrate mutagenic potential in these in vitro cell mutation assays.

Chromosomal aberration:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, cultured human lymphocytes were exposed to the test item.

Human lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin (PHA), and exposed to the test substance both in the absence and presence of S9 mix derived from rat livers. Vehicle and positive control cultures were also included. Two hours before the end of the incubation period, cell division was arrested using Colcemid®, the cells harvested and slides prepared, so that metaphase cells could be examined for chromosomal damage.

A preliminary toxicity test was performed in order to determine the toxicity of Terpenic oligomers to cultured human lymphocytes. A 3-hour treatment in the absence and presence of S9 mix, and a 21-hour continuous treatment in the absence of S9 mix were used to determine toxicity.

In the main test, the mitotic index was assessed for all cultures treated with Terpenic oligomers and the vehicle control, acetone. Justification for the highest analysed concentration was based on the highest final concentration for this test system, where no change of greater than 50 mOsmol/kg when compared to the concurrent vehicle control. Otherwise, selection was determined by cytotoxicity.

On the basis of these data, the following concentrations were selected for metaphase analysis:

- In the absence of S9 mix, 3-hour treatment: 750, 1000 and 1250 μg/mL.

- In the presence of S9 mix, 3-hour treatment: 250, 1000 and 1250 μg/mL / Additional test: 750, 1000 and 1250 μg/mL.

- In the absence of S9 mix, 21-hour continuous treatment: 31.25, 250 and 500 μg/mL.

In the absence and presence of S9 mix following 3-hour treatment, Terpenic oligomers caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations, at any analysed concentration, when compared to the vehicle control. Same results were obtained In the absence of S9 mix following 21-hour continuous treatment.

No statistically significant increases in the proportion of polyploid or endoreduplicated metaphase cells were observed during metaphase analysis, under any treatment condition, when compared to the vehicle control.

All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. Both positive control compounds caused statistically significant increases in the proportion of aberrant cells, demonstrating the sensitivity of the test system and the efficacy of the S9 mix.

Therefore, test item Terpenic oligomers has shown no evidence of causing an increase in the frequency of structural chromosome aberrations in this in vitro cytogenetic test system.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to CLP Regulation (EC) No. 1272/2008 and to the UN GHS Regulation.