Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Farnesane is not mutagenic per the Ames assay (OECD 471) as conducted with E. coli WP2 uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100 (MB Research Laboratories, 2010).

Farnesane, also did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells in a Guideline OECD 476 study (Harlan Laboratories, 2014b) and was considered to be non mutagenic under the conditions of the test.

A OECD Guideline 473 study was also conducted to assess the potential for chromosomal aberrations (BioReliance, 2013). Farnesane was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non activated test system in the in vitro mammalian chromosome aberration test using human peripheral blood lymphocytes. In the S9-activated test system, Farnesane was clastogenic at 100 µg/mL.  At this dose, precipitate was observed at the end of the treatment period, and neat test article was present throughout the treatment period. Cells were therefore exposed to the neat test article and not to a specific concentration of the test article which creates a micro-environment where some cells are acutely exposed to a relatively higher concentration of test article. This raises the strong possibility that the low level of clastogenicity observed at 100 µg/mL may be associated with the test article precipitate and is therefore not considered biologically relevant (Kirkland and Muller, 2000).

In their decision letter dated November 2016 (Decision number: CCH-D-2114347533-50-01/F - provided as an attachment in Section 13 of the dossier), ECHA noted that the water solubility of the substance measured for the chromosomal aberration test (i.e. 50 mg/mL) was not consistent with the physico chemical data provided in the registered dossier for this substance (in IUCLID section 4.8. (Water solubility), it is stated that the ‘water solubility of Farnesane is very low and between 0.25 µg/L to 4.4 µg/L). Farnesane is therefore considered essentially insoluble in water.’ Compared to these data, the water solubility mentioned for the chromosomal aberration test was seven orders of magnitude higher. It was unclear to ECHA how this discrepancy in water solubility could be explained, and how concentrations as high as 50 or 100 µg/mL could be achieved in the chromosomal aberration assay by using water as a solvent.

 

In ECHA’s view, since the test substance is a colourless liquid, the lack of solubility may not have been detected using the “visual solubility test” performed by the laboratory (BioReliance, 2013). ECHA acknowledged that the OECD TG 473 says “liquid test substances may be added directly to the test systems and/or diluted prior to treatment”. However, according to the physico-chemical data provided in the dossier, the substance cannot be diluted in water or aqueous media to obtain an acceptable concentration.

 

The existing in vitro chromosomal aberration study was therefore considered unreliable (and downgraded; Klimisch Score = 3) since the test was conducted in an aqueous test system and the test material is essentially insoluble in water. The other in vitro genotoxicity studies were undertaken using organic solvent systems (ethanol and acetone), which are at variance with the system used in the chromosome aberration study.

Based on the information provided above, an additional OECD Guideline 473 in vitro chromosome aberration study (Covance, 2018a) was conducted using DMF as the solvent vehicle. In this study, farnesane did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes in both the absence and presence of S-9 when tested up to 2000 µg/mL, an acceptable maximum concentration for in vitro chromosome aberration studies according to current regulatory guidelines, and/or up to precipitating concentrations.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 18-November 24, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Follows OECD guidelines and in accordance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Characteristics of Tester Strains

Tester Strain Gene Affected
E. coli WP2 uvrA T/PE


S. typh. TA-97a his D 6610

S. typh. TA-1535 his G 46

S. typh. TA-98 his D 3052

S. typh. TA-100 his G 46
Species / strain / cell type:
bacteria, other:
Additional strain / cell type characteristics:
other: See "Target Gene"
Metabolic activation:
with and without
Metabolic activation system:
S9 (Mitochondrial supernatant from liver of Sprague Dawley® rat induced by Aroclor 1254 )
Test concentrations with justification for top dose:
Based on the cytotoxicity results, five concentrations (50, 100, 500, 1000 and 5000 nl/plate) of the test article were tested in each of five bacterial tester strains (E. coli WP2 uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: 95% ethanol;
- Justification for choice of solvent/vehicle: Prior to the cytotoxic screen, solubility of the test article was checked in tissue culture water, Dimethyl sulfoxide, 95% Ethanol (EtOH) and Acetone. The test article was freely soluble at a concentration of 50 µl/ml in 95% EtOH and Acetone. The Sponsor, in consultation with the Study Director, chose 95% ethanol as the vehicle for the assay.
The test article did not mix well with the top agar in the plates at 1000 and 5000 nl/plate, with and without S9. However, vortexing the test article/agar immediately prior to plating produced a useable mixture.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA); Methyl methanesulfonate (MMS); Acridine, 6-chloro-9-(3-((2-chloroethyl)amino) propyl)amino-2-methoxy, dihydrochloride (ICR-191); Sodium azide (NaN3); Daunomycin hydrochloride (DM)
Details on test system and experimental conditions:
DURATION
- Preincubation period: overnight cultures were inoculated by the addition of a lyophilized disk of each tester strain to Oxoid No.2 nutrient broth. Ampicillin was added to the nutrient broth to ensure the retention of R-factor plasmid in tester strains TA-97a, TA-98 and TA-100. The cultures were incubated at 37ºC ±2ºC with agitation. The cultures were used after they reach the late exponential growth phase as determined by absorbance readings at 600 nm.

- Exposure duration: 48-72 hours. Top agar supplemented with appropriate amino acids were prepared, as 2 ml aliquots, and maintained at 45-50ºC in sterile culture tubes. Dulbecco’s Phosphate Buffered Saline (DPBS) was added to the tubes not undergoing S9 activation (i.e. without S9, or –S9) to maintain equal dosing volumes. 0.1 ml of bacteria was added to the top agar, followed by 0.1 ml of the test article, vehicle control or positive control. For the activation portion of the test, 0.5 ml of S9 mixture was added last. The contents were vortexed and overlaid onto minimal glucose agar plates. After the mixture had solidified, the plates were incubated at 37ºC ± 2ºC for 48-72 hours. Plates that were not scored immediately following the incubation period were stored at 2-8ºC until scoring.

- Expression time (cells in growth medium): 48 - 72 hours

-Revertant Colony Count:
Counting of the revertants per plate was performed using an AlphaImager™ 2200 (Alpha Innotech Corporation, San Leandro, CA) fluorescence imager. Proper function of the imager was verified against a standard template (e.g. high (1000), medium (100) and low (10) counts) prior to each daily use. The number of revertants was recorded, along with observations of cytotoxicity. Routine examination (under a light microscope) of the bacterial background lawn was used to determine cytotoxicity of the test article. The plates were also examined visually for test article precipitate.

NUMBER OF REPLICATIONS: Five concentrations (50, 100, 500, 1000 and 5000 nl/plate) of the test article were tested in each of five bacterial tester strains. Two sets of culture plates were dosed per concentration (+S9 and No S9). A vehicle control and positive controls specific to each bacterial strain were treated in the same manner as the test article concentrations.
Evaluation criteria:
Plates were scored based on the number of revertant colony-forming units present per plate. The number of revertants of each test article plate were averaged and plotted versus concentration of the test article. The mean number of revertants of each dose was divided by the mean for the vehicle control value to obtain a ratio to vehicle. In evaluating the data, cytotoxicity of the test article as well as quality checks of the assay were taken into account 3,4.

In general, a 2-fold increase with or without metabolic activation is considered a positive response. Dose-related increases approaching a 2-fold increase are deemed equivocal.

A negative result is determined by the absence of a dose-related increase in all five tester strains, again taking into account cytotoxicity of the test article as well as the quality checks of the assay.

Positive results from the bacterial reverse mutation test indicate that the substance induces point mutations by base substitutions or frame shifts in the genome of either Salmonella typhimurium and/or Escherichia coli. Negative results indicate that under the test conditions, the test substance is not mutagenic in the tested species.
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
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
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
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 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None identified
- Effects of osmolality: None identified
- Evaporation from medium: None identified
- Water solubility: See "other confounding effects"
- Precipitation: The plates were also examined visually for test article precipitate. none identified
- Other confounding effects: The test article did not mix well with the top agar in the plates at 1000 and 5000 nl/plate, with and without S9. However, vortexing the test article/agar immediately prior to plating produced a useable mixture. There was no diminution or clearing of the background lawn observed at any of the dosages, and the number of revertants from test article treatments approximated that of the vehicle (95% Ethanol) control. The results showed that the test article was not cytotoxic to TA100 at 1 to 5000 ug/plate. In the main study, 5000 nl/plate was chosen as the top concentration for the test article.

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA:

Vehicle Control
The spontaneous reversion rate, as represented by the mean colony forming units (CFU), for each strain of bacteria was measured and compared to in-house historical ranges. All vehicle controls passed the quality check.

Mean CFU Mean CFU per Control – Historical Range
Tester Strain +S9 –S9 +S9 –S9
E. coli WP2uvrA 35.2 35.7 14-90 14-115
S. typh. TA-97a 65.0 56.7 33-211 22-149
S. typh. TA-1535 10.5 8.8 3-26 2-36
S. typh. TA-98 47.0 46.2 7-85 7-84
S. typh. TA-100 72.8 69.3 51-255 56-239

Positive Control
The increase in revertants due to positive control treatment for each tester strain of bacteria was calculated.
All positive controls passed the quality check..

Mean CFU Fold Increase over Vehicle Control
Tester Strain +S9 –S9 +S9 –S9
E. coli WP2uvrA 193.2 547.3 5.5  15.3 
S. typh. TA-97a 601.5 1009.3 9.3  17.8 
S. typh. TA-1535 155.0 468.8 14.8  53.3 
S. typh. TA-98 2694.0 1132.2 57.3  24.5 
S. typh. TA-100 2419.5 486.7 33.2  7.0 

 = 2-fold or more increase over vehicle control

Sterility Test
Solutions and reagents used in the assay were tested for sterility in the main assay. Samples were added to minimal glucose agar plates and incubated at 37ºC ±2ºC for approximately 72 hours. No contaminating microorganisms were detected in any of the reagents used in the assay.

Bacterial Growth Observed
Component Main Test
Top Agar with L-Tryptophan None
Top Agar with L-Histidine and D-Biotin None
Test Article at 50 µg/plate None
95% EtOH None
DPBS None
Nutrient Broth None
10% S9 None

The sterility test passed the quality check.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No reduced or clearing bacterial background lawn was observed, indicating no or minimal cytotoxicity of the test article under the test conditions.

The assay was run in all five strains on triplicate plates. Positive and vehicle controls were run concurrently for all five strains, on six plates per strain. All plating was with and without exogenous metabolic activation. Heterogeneity of the test article in plates at 1000 and 5000 nl/plate, with and without S9, in all bacterial strains did not interfere the automatic colony counting. No reduced or clearing bacterial background lawn was observed, indicating no or minimal cytotoxicity of the test article under the test conditions. There is neither significant increase nor dose-dependent increase of the number of revertants in any bacterial strain treated with the test article in the presence or absence of S9. All positive and negative control values were within acceptable ranges, and all criteria for a valid study were met.

See "Attached background material" section below

Conclusions:
Interpretation of results: negative

Farnesane is not mutagenic per the Ames assay as conducted with E. coli WP2 uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100
Executive summary:

Objective: The purpose of this study is to evaluate the mutagenic potential of a test article based on the reversion of selective growth mutations in several strains of Salmonella typhimurium bacteria and in Escherichia coli WP2 uvrA bacteria, in the presence and absence of S9 activation. This protocol is based on OECD Guideline for Testing of Chemicals: No. 471 – Bacterial Reverse Mutation Test and U.S. EPA Health Effects Test Guidelines OPPTS 870.5100 – Bacterial Reverse Mutation Test.

 

Method Synopsis: Prior to the cytotoxic screen, solubility of the test article was checked in tissue culture water, Dimethyl sulfoxide, 95% Ethanol (EtOH) and Acetone. The test article was freely soluble at a concentration of 50 µL/mL in 95% EtOH and Acetone. The Sponsor, in consultation with the Study Director, chose 95% ethanol as the vehicle for the assay. A cytotoxicity screen was conducted in the TA-100 tester strain using eight concentrations (1, 5, 10, 50, 100, 500, 1000 and 5000nl/plate) of the test article, two plates per dose,on the bacterial tester strain Salmonella typhimurium TA-100. The test article was combined with the bacteria and top agar in the presence and absence of a metabolic activation mixture (S9) and overlain onto minimal glucose agar plates. A 95% EtOH vehicle control was run concurrently, with and without S9.

 

Based on the cytotoxicity results, five concentrations (50,100,500,1000 and 5000nl/plate) of the test article were tested in each of five bacterial tester strains (E. coli WP2uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100).  Vehicle controls and positive controls specific to each bacterial strain were treated in the same manner as the test article concentrations. The plates were incubated at 37ºC ± 2ºC for 48-72 hours. Revertant colony growth was determined by counting the colonies per plate using an AlphaImager™imaging system. The number of revertants of the test article treatment plates and positive control plates was divided by the number of revertants of the vehicle plates. In general, a positive result is determined by a 2-fold increase above the vehicle control.

  

Summary: Farnesane in the vehicle, Ethanol, was tested in a Bacterial Reverse Mutation Assay. The test article did not show obvious cytotoxicity to bacterial strain TA100 at dose range 1 to 5000 nl/plate, with or without S9. The test article at 50 to 5000 nl/plate, with or without S9, did not cause significant increase or dose-dependent increase in the number of revertants of any bacterial tester strains. This indicates that the test article is negative for mutagenicity in the Bacterial Reverse Mutation Assay. 

 

Conclusion: Under test conditions, test article farnesane does not have mutagenicity potential.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-05-31 to 2018-09-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Amyris, Inc.; Batch no. 17GLY03173080182
- Expiration date of the lot/batch: 2020-03-17
- Purity test date: 2017-03-17

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at 15-25°C, protected from light
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in dimethylformamide (DMF) at concentrations up to at least 211.32 mg/mL

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Clear liquid

OTHER SPECIFICS:
Purity: 99%
molecular weight: 212.421 g/mol
Species / strain / cell type:
lymphocytes: cultured human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Blood from three healthy, non-smoking female volunteers from a panel of donors at Covance was used for each experiment (See Table 2)
- Suitability of cells: The use of human peripheral blood lymphocytes is recommended because the cells are only used in short-term culture and maintain a stable karyotype (Evans & O’Riordan, 1975). Experiments with these cells can also be performed in conjunction with S-9 since, for short incubation periods, no toxicity is induced by the liver homogenate itself.
- Cell cycle length, doubling time or proliferation index: Cell cycle length: 13 ± 2 hours
- Sex, age and number of blood donors if applicable: 3 females (for age see Table 2)
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Methods for maintenance in cell culture if applicable: For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within two days of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.

Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 8.5 mL (Range-Finder and 3+21 hour treatments in the absence and presence of S-9) or 8.55 mL (20+0 hour treatments in the absence of S-9, Trial 2) pre-warmed (in an incubator set to 37±1°C) HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin, so that the final volume following addition of S-9 mix or KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37±1°C for approximately 48 hours and rocked continuously.
- Modal number of chromosomes: 44-48.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin, so that the final volume following addition of S-9 mix or KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not specified
- Periodically checked for karyotype stability: Not specified
- Periodically 'cleansed' against high spontaneous background: Not specified
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Prepared from male Sprague Dawley rats induced with Aroclor 1254 (Molecular Toxicology Incorporated, USA)
Test concentrations with justification for top dose:
A maximum concentration of 2000 µg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to the recommended maximum concentration for testing pharmaceuticals in in vitro chromosome aberration studies (OECD, 2016). Concentrations for the Chromosome Aberration Experiment were selected based on the results of this cytotoxicity Range-Finder Experiment (See Table 1).

Range-Finder (3+17 Hour Treatments, ±S-9): 7.256, 12.09, 20.16, 33.59, 55.99, 93.31, 155.5, 259.2, 432.0, 720.0, 1200, 2000 µg/mL

Range-Finder (20+0 Hour Treatments, -S-9): 7.256, 12.09, 20.16, 33.59, 55.99, 93.31, 155.5, 259.2, 432.0, 720.0, 1200, 2000 µg/mL

Experiment Trial 1 (3+17 Hour Treatments, +S-9): 0, 200, 400, 800, 1200, 1600, 2000 µg/mL

Experiment Trial 2 (20+0 Hour Treatments, -S-9): 0, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1600, 2000 µg/mL

Experiment Trial 3 (3+17 Hour Treatments, -S-9): 0, 200, 400, 800, 1200, 1400, 1600, 1800, 2000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethylformamide (DMF)

- Justification for choice of solvent/vehicle: Preliminary solubility data indicated that 2,6,10-Trimethyldodecane was soluble in dimethylformamide (DMF) at concentrations up to at least 211.32 mg/mL. The solubility limit in culture medium was in the range of 132.075 to 264.15 µg/mL, as indicated by precipitation at the higher concentration which persisted for at least 24 hours after test article addition.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Blood cultures were incubated at 37±1°C for approximately 48 hours and rocked continuously.
- Exposure duration: See Table 1 for details
- Expression time (cells in growth medium): See Table 1 for details
- Fixation time (start of exposure up to fixation or harvest of cells): Approximately 2 hours prior to harvest, colchicine was added to give a final concentration of approximately 1 µg/mL to arrest dividing cells in metaphase. At the defined sampling time cultures were centrifuged at approximately 300 g for 10 minutes; the supernatant was carefully removed and cells were resuspended in 4 mL pre-warmed hypotonic (0.075 M) KCl, and incubated at 37±1ºC for 15 minutes to allow cell swelling to occur. Cells were fixed by dropping the KCl suspension into fresh, cold methanol/glacial acetic acid (3:1, v/v). The fixative was changed by centrifugation (approximately 300 g, 10 minutes) and resuspension. This procedure was repeated as necessary (centrifuging at approximately 1250 g, two to three minutes) until the cell pellets were clean.

SPINDLE INHIBITOR (cytogenetic assays): colchicine

STAIN (for cytogenetic assays): filtered 4% (v/v) Giemsa in pH 6.8 Gurr’s buffer

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Lymphocytes were kept in fixative at 2-8ºC prior to slide preparation for a minimum of 3 hours to ensure that cells were adequately fixed. Cells were centrifuged (approximately 1250 g, two to three minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of 45% (v/v) aqueous acetic acid were added to each suspension to enhance chromosome spreading, and several drops of suspension were transferred on to clean microscope slides labelled with the appropriate study details. Slides were flamed, as necessary, to improve quality. Slides were dried on a hot plate (set to approximately 80-100°C) then stained in filtered 4% (v/v) Giemsa in pH 6.8 Gurr’s buffer for 5 minutes. The slides were rinsed, dried and mounted with coverslips using DPX.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): A minimum of 300 metaphases per concentration (150 metaphases from each code) were analysed for chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
Rationale for test conditions:
The purpose of the in vitro chromosome aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells. Structural aberrations may be of two types, chromosome or chromatid. Polyploidy (including endoreduplication) could arise in chromosome aberration assays in vitro. However, while aneugens can induce polyploidy, polyploidy alone does not indicate aneugenic potential and may simply indicate cell cycle perturbation or cytotoxicity (Muehlbauer et al., 2008). As such, although numerical aberrations can be detected, this assay is not specifically designed for this type of evaluation.

The use of human peripheral blood lymphocytes is recommended because the cells are only used in short-term culture and maintain a stable karyotype (Evans & O’Riordan, 1975). Experiments with these cells can also be performed in conjunction with S-9 since, for short incubation periods, no toxicity is induced by the liver homogenate itself.

In the Chromosome Aberration Experiment, cells were exposed to the test article both in the absence and presence of S-9 (from rats induced with Aroclor 1254 (Maron & Ames, 1983)) for 3 hours and sampled at 20 hours after the beginning of treatment. This is equivalent to approximately one and a half times the average generation time of cultured lymphocytes from the panel of donors used in this laboratory. As a number of chemicals have been reported as only exerting positive effects following prolonged treatment (Ishidate, 1987; Galloway et al., 1987; Galloway et al., 1994), cells were also exposed to the test item continuously for 20 hours in the absence of S 9.

As data from the initial experiment could not be clearly identified as either negative or positive and following consultation with the Study Monitor, a second experiment was performed for 20+0 and 3+17 hour treatments in the absence of S-9.
Evaluation criteria:
For valid data, the test article was considered to induce clastogenic events if:

1. A statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) at one or more concentrations was observed (p ≤0.05)
2. The incidence of cells with structural aberrations (excluding gaps) at such a concentration that exceeded the normal range in both replicate cultures
3. A concentration-related increase in the proportion of cells with structural aberrations (excluding gaps) was observed (positive trend test).

The test article was considered positive in this assay if all of the above criteria were met.

The test article was considered negative in this assay if none of the above criteria were met.

Results which only partially satisfied the above criteria were to be dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance was taken into account, for example consistency of response within and between concentrations.
Statistics:
After completion of scoring and decoding of slides the numbers of aberrant cells in each culture were categorized as follows:

1. Cells with structural aberrations including gaps
2. Cells with structural aberrations excluding gaps
3. Polyploid or endoreduplicated cells.

The totals for category 2 in vehicle control cultures were compared with the current historical vehicle control (normal) ranges to determine whether the assay was acceptable or not (see Acceptance criteria). The proportion of cells with structural chromosome aberrations excluding gaps were compared with the proportion in vehicle controls by using Fisher’s exact test (Richardson et al., 1989). In addition, a Cochran-Armitage Trend Test was performed to aid determination of concentration response relationships. Probability values of p≤0.05 were accepted as significant. The proportions of cells in categories 1 and 3 were examined in relation to historical vehicle control range.

The proportions of aberrant cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989). Probability values of p≤0.05 were accepted as significant.
Key result
Species / strain:
lymphocytes: human peripheral blood lymphocytes
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH; Effects of osmolality: No marked changes in osmolality (of greater than 50 mOsm/kg) or pH (of greater than one unit) were observed at the highest concentration tested in the Range-Finder Experiment (2000 µg/mL) as compared to the concurrent vehicle controls
- Evaporation from medium: not specified
- Precipitation: precipitation at the end of treatment was not observed in the Range Finder Experiment. Further details are presented in Table 4.
- Definition of acceptable cells for analysis: Only cells with 44 to 48 chromosomes were considered acceptable for analysis. Any cell with more than 48 chromosomes (that is, polyploid or endoreduplicated cells) observed during this evaluation was noted and recorded separately

RANGE-FINDING/SCREENING STUDIES: No marked changes in osmolality (of greater than 50 mOsm/kg) or pH (of greater than one unit) were observed at the highest concentration tested in the Range-Finder Experiment (2000 µg/mL) as compared to the concurrent vehicle controls (individual data not reported).
 
The results of the MI determinations from the cytotoxicity Range-Finder Experiment were as presented in Tables 7, 8, and 9. The results of the cytotoxicity Range-Finder Experiment were used to select suitable maximum concentrations for the Chromosome Aberration Experiment, Trial 1.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: See Table 6.
- Negative (solvent/vehicle) historical control data: See Table 5.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Mitotic Index (See Table 4).

Table 4. Results - summary of the chromosome aberration data

Treatment

Concentration (mg/mL)

Cytotoxicity (%)$

% Cells with Chromosome Aberrations (Excluding Gaps)

Historical Control Range
(%)#

Statistical Significance

3+17 hour

-S-9

Vehiclea

-

1.00

0 – 2.67

-

Trial 3

200.0

4

0.33

 

NS

800.0

0

0.33

 

NS

1200

2 ^

1.33

 

NS

2000

0 ^

0.33

 

NS

*MMC, 0.30

6

25.44

 

p≤0.001

3+17 hour

+S-9

Vehiclea

-

0.00

0 – 3.33

-

Trial 1

400.0

8

0.33

 

NS

1200

24

0.00

 

NS

2000

28

1.00

 

NS

*CPA, 4.00

43

9.00

 

p≤0.001

20+0 hour

-S-9

Vehiclea

-

0.33

0 – 1.33

-

Trial 2

100.0

23

1.00

 

NS

500.0

7

0.67

 

NS

800.0

31

0.00

 

NS

900.0

17 ^

0.00

 

NS

*MMC, 0.10

47

32.97

 

p≤0.001

 a         Vehicle control was DMF

*          Positive control

#         Observed range

$         Based on mitotic index

^          Precipitation observed at the end of treatment incubation

NS       Not Significant

Historical Vehicle Control Ranges

Data generated from studies performed within the GLP laboratory, by GLP trained staff, whether a claim of GLP compliance was made or not, were included in the compilation of the historical control ranges without bias.

 

Table 5. Historical Vehicle Control Ranges – Female Donors

 

Structural Aberrations Frequency

Numerical Aberrations Frequency

Structural Aberrations
Including Gaps

Structural Aberrations
Excluding Gaps

Endo reduplicated Cells

Polyploid Cells

%

%

%

%

3+17, -S9

Number of experiments

7

Number of cultures

32

Mean

0.42

0.29

0.0

0.04

Standard deviation

0.81

0.72

0.0

0.24

Observed range

0.0 - 2.67

0.0 - 2.67

0.0 - 0.0

0.0 - 1.33

95% Reference range

-

-

-

-

3+17, +S9

Number of experiments

6

Number of cultures

28

Mean

0.74

0.5

0.0

0.02

Standard deviation

0.84

0.8

0.0

0.13

Observed range

0.0 - 3.33

0.0 - 3.33

0.0 - 0.0

0.0 - 0.67

95% Reference range

-

-

-

-

20+0, -S9

Number of experiments

6

Number of cultures

28

Mean

0.52

0.17

0.0

0.0

Standard deviation

0.9

0.39

0.0

0.0

Observed range

0.0 - 2.67

0.0 - 1.33

0.0 - 0.0

0.0 - 0.0

95% Reference range

-

-

-

-

Reference ranges are not appropriate for ranges with few than 40 cultures.

Ranges calculated in April 2018 from experiments started between June 2015 and June 2017.

Historical Positive Control Ranges

Data generated from studies performed within the GLP laboratory, by GLP trained staff, whether a claim of GLP compliance was made or not, were included in the compilation of the historical control ranges without bias.

Table 6. Historical Positive Control Ranges – Female Donors

 

Structural Aberrations Frequency

Numerical Aberrations Frequency

Structural Aberrations
Including Gaps

Structural Aberrations
Excluding Gaps

Endo reduplicated Cells

Polyploid Cells

%

%

%

%

3+17,
-S9

MMC,
0.30 µg/mL

Number of experiments

4

Number of cultures

16

Mean

21.76

20.91

0.0

0.0

Standard deviation

20.08

20.49

0.0

0.0

Observed range

6.0 - 88.24

5.33 - 88.24

0.0 - 0.0

0.0 - 0.0

95% Reference range

-

-

-

-

3+17,
+S9

CPA,
1.0 µg/mL

Number of experiments

4

Number of cultures

15

Mean

8.31

7.42

0.0

0.0

Standard deviation

2.62

1.92

0.0

0.0

Observed range

5.33 - 16.0

5.33 - 12.0

0.0 - 0.0

0.0 - 0.0

95% Reference range

-

-

-

-

CPA,
2.0 µg/mL

Number of experiments

2

Number of cultures

4

Mean

26.05

23.97

0.0

0.0

Standard deviation

9.13

9.19

0.0

0.0

Observed range

19.33 - 39.02

16.67 - 36.59

0.0 - 0.0

0.0 - 0.0

95% Reference range

-

-

-

-

20+0,
-S9

MMC,
0.10 µg/mL

Number of experiments

3

Number of cultures

10

Mean

20.94

19.48

0.0

0.0

Standard deviation

10.56

11.11

0.0

0.0

Observed range

11.33 - 48.39

9.33 - 48.39

0.0 - 0.0

0.0 - 0.0

95% Reference range

-

-

-

-

Reference ranges are not appropriate for ranges with few than 40 cultures.

Ranges calculated in April 2018 from experiments started between June 2015 and June 2017.

Table 7. Data for 3+17 Hour Treatments, -S-9, Range-Finder – Female Donors

Treatment (µg/mL)

Replicate

Mitotic Index (%)

MIH * (%)

Vehicle

A

13.6

 

 

B

10.9

 

 

Total

12.3

-

7.256

A

13.0

0

12.09

A

10.8

12

20.16

A

10.2

17

33.59

A

11.7

4

55.99

A

10.8

12

93.31

A

10.2

17

155.5

A

11.0

10

259.2

A

9.7

21

432.0

A

10.7

13

720.0

A

8.3

32

1200

A

10.8

12

2000

A

10.2

17

Table 8. Data for 3+17 Hour Treatments, +S-9, Range-Finder – Female Donors

Treatment (µg/mL)

Replicate

Mitotic Index (%)

MIH * (%)

Vehicle

A

10.0

 

 

B

12.0

 

 

Total

11.0

-

7.256

A

12.2

0

12.09

A

11.3

0

20.16

A

10.9

1

33.59

A

9.2

16

55.99

A

9.3

15

93.31

A

9.4

15

155.5

A

9.6

13

259.2

A

11.2

0

432.0

A

13.4

0

720.0

A

9.8

11

1200

A

10.7

3

2000

A

10.9

1

* Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100%

(where T = treatment and C = negative control)

 

Table 9. Data for 20+0 Hour Treatments, -S-9, Range-Finder – Female Donors

Treatment (µg/mL)

Replicate

Mitotic Index (%)

MIH * (%)

Vehicle

A

5.0

 

 

B

2.9

 

 

Total

4.0

-

7.256

A

4.6

0

12.09

A

3.1

22

20.16

A

3.8

4

33.59

A

4.4

0

55.99

A

3.6

9

93.31

A

3.7

6

155.5

A

3.4

14

259.2

A

2.4

39

432.0

A

1.6

59

720.0

A

4.0

0H

1200

A

NM

- H

2000

A

NM

- H

H = Indicates precipitation observed at harvest

* Mitotic inhibition (%) = [1 - (mean MIT/mean MIC)] x 100%

(where T = treatment and C = negative control)

Validity of Study

The results data confirm that:

1.    The binomial dispersion test demonstrated acceptable heterogeneity between replicate cultures following treatments in the absence of S-9. Significant(p≤0.05) heterogeneity was noted for 3+17 hour +S-9 treatments, however, as the chromosome aberration data was clearly negative, the heterogeneity was considered not relevant

2.    The proportion of cells with structural aberrations (excluding gaps) in vehicle control cultures fell within the normal range.

3.    At least 300 cells were suitable for analysis at each concentration, unless 15 or more cells showing structural aberrations (per slide) other than gaps only were observed during analysis.

4.    The positive control chemicals induced statistically significant increases in the proportion of cells with structural aberrations. Both replicate cultures at the positive control concentration analyzed under each treatment condition demonstrated structural aberration cell frequencies (excluding gaps) that clearly exceeded the current historical vehicle control ranges

5.    The maximum concentration analysed under each treatment condition was the maximum concentration tested or the lowest concentration at which precipitate was observed at the end of the treatment period.

Structural Aberrations

Treatment of cells with 2,6,10-Trimethyldodecanefor 3+17 hours in the absence and presence of S-9 and for 20+0 hours in the absence of S-9 resulted in frequencies of cells with structural chromosome aberrations which were similar to and not significantly higher than those observed in concurrent vehicle control cultures for all concentrations analyzed. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within the normal ranges.

Numerical Aberrations

Sporadic increases in polyploidy cells in cultures treated with 2,6,10-Trimethyldodecane, which exceeded the normal range, were observed at the lowest and highest concentration analyzed following 3+17 hour treatment in the presence of S-9. However, these marginal increases were also observed in the vehicle control and positive control cultures. Furthermore, numerical aberrations were not scored quantitatively. As such these sporadic increases can be considered of no biological relevance.

Conclusions:
It was concluded that 2,6,10-Trimethyldodecane did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes in both the absence and presence of S-9 when tested up to 2000 µg/mL, an acceptable maximum concentration for in vitro chromosome aberration studies according to current regulatory guidelines, and/or up to precipitating concentrations.
Executive summary:

In a key OECD Guideline 473 in vitro chromosome aberration assay, the clastogenic potential of the test material (2,6,10-Trimethyldodecane) was tested using duplicate human lymphocyte cultures prepared from the pooled blood of three female donors in a single experiment conducted across three trials. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in dimethylformamide (DMF). A preliminary cytotoxicity Range-Finder Experiment was performed up to 2000 µg/mL (an acceptable maximum concentration for in vitro chromosome aberration studies according to current regulatory guidelines), and appropriate concentrations were determined for the Chromosome Aberration Experiment.

 

Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for chromosome analysis were selected by evaluating the effect of 2,6,10-Trimethyldodecane on mitotic index. Chromosome aberrations were analyzed at three or four concentrations.

 

Appropriate negative (vehicle and untreated) control cultures were included in the test system in the Chromosome Aberration Experiment under each treatment condition (untreated control included for 20+0 hour –S-9 treatments only). The proportion of cells with structural aberrations in vehicle control cultures fell within current observed historical vehicle control (normal) ranges. It was therefore not considered necessary to analyze the untreated control cultures. Mitomycin C (MMC) and cyclophosphamide (CPA) were employed as positive control chemicals in the absence and presence of rat liver S-9 respectively. Cells receiving these were sampled 20 hours after the start of treatment; both compounds induced statistically significant increases in the proportion of cells with structural aberrations.

 

All acceptance criteria were considered met and the study was therefore accepted as valid. Treatment of cells with 2,6,10-Trimethyldodecane for 3+17 hours in the absence and presence of S-9 and for 20+0 hours in the absence of S-9 resulted in frequencies of cells with structural chromosome aberrations which were similar to and not significantly higher than those observed in concurrent vehicle control cultures for all concentrations analyzed. Numbers of aberrant cells (excluding gaps) in all treated cultures fell within the normal ranges.

 

Sporadic increases in polyploidy cells in cultures treated with 2,6,10-Trimethyldodecane, which exceeded the normal range, were observed at the lowest and highest concentration analyzed following 3+17 hour treatment in the presence of S-9. However, these marginal increases were also observed in the vehicle control and positive control cultures. Furthermore numerical aberrations were not scored quantitatively. As such these sporadic increases can be considered of no biological relevance.

 

It was concluded that 2,6,10-Trimethyldodecane did not induce structural chromosome aberrations in cultured human peripheral blood lymphocytes in both the absence and presence of S-9 when tested up to 2000 µg/mL, an acceptable maximum concentration for in vitro chromosome aberration studies according to current regulatory guidelines, and/or up to precipitating concentrations.

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:
12 February 2014 to 24 April 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Type and identity of media:
RPMI 1640 (R0)

Properly maintained:
Yes

Periodically checked for Mycoplasma contamination:
Yes

Periodically checked for karyotype stability:
No

Periodically "cleansed" against high spontaneous background:
Yes

Date of GLP inspection: 12 - 14 March 2014 Date of GLP signature: 12 May 2014
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
The maximum dose level used was limited by the onset of greasy / oily precipitate effectively reducing exposure of the test item to the cells. Vehicle and positive controls were used in parallel with the test item. Solvent (Acetone) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch BCBK5968V at 400 µg/mL and 150 µg/mL for Experiment 1 and Experiment 2, respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0302605 at 2 µg/mL and 1.7 µg/mL for Experiment 1 and Experiment 2, respectively, was used as the positive control in the presence of metabolic activation. The positive controls were formulated in DMSO.
Vehicle / solvent:
Following solubility checks performed in-house, the test item was accurately weighed and formulated in acetone prior to serial dilutions being prepared.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (Acetone) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Cyclophosphamide
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Solvent (Acetone) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
This study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

The use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci. The thymidine kinase heterozygote system, TK +/- to TK -/-, was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This system has been extensively validated (Clive et al., 1979; Amacher et al., 1980; Jotz and Mitchell, 1981).

The technique used was a fluctuation assay using microtitre plates and trifluorothymidine as the selective agent and is based on that described by Cole and Arlett (1984). Two distinct types of mutant colonies can be recognized, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity)

Guidelines / Regulations

This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:

• OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Test" adopted 21 July 1997
• Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008
• US EPA OPPTS 870.5300 Guideline

The test method is designed to be in alignment with the following Japanese Guidelines

• ``Kanpoan No. 287 - - Environment Protection Agency``
• ``Eisei No. 127 - - Ministry of Health and Welfare``
• ``Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry``
Evaluation criteria:
Please see "Any other information on materials and methods incl. tables" section.
Statistics:
Please see "Any other information on materials and methods incl. tables" section.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 4.15 to 1062 µg/mL, the upper dose level was the maximum achievable dose level. There was no evidence of any reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle control in the 4-hour exposure group in the presence of metabolic activation. However, a very modest decrease was observed in the 4-hour exposure group in the absence of metabolic activation, and a greater decrease was observed in the 24-hour exposure group in the absence of metabolic activation. Precipitate of the test item was observed at and above 66.38 µg/mL in all three of the exposure groups and became greasy / oily in appearance overall at and above 132.75 µg/mL. Based on the %RSG values observed, and the onset of precipitate effectively reducing exposure of the test item to the cells as indicated by the %RSG values, maximum exposure of the test item to the cells was considered to occur at approximately 33.19 to 66.38 µg/mL. The maximum dose levels in the subsequent mutagenicity test were therefore set at 68 µg/mL.

Mutagenicity Test

A summary of the results from the test is presented in attached Table 1.

Experiment 1

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.

The formulation analysis on the highest dose concentration of Experiment 1 demonstrated that the dose formulation was within the required parameter of less than a 10% error, on this occasion there was a 1% error, Determination of Content, Homogeneity and Stability of Test Item in Application Formulations Report attached.

As was seen in the preliminary toxicity test, there was no evidence of any marked toxicity following exposure to the test item in either the absence or presence of metabolic activation, as indicated by the %RSG and RTG values (Tables 3 and 6). There was also no evidence of any significant reductions in viability (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred in either of the exposure groups (Tables 3 and 6). The toxicity observed in both the absence and presence of metabolic activation was similar to that seen in the preliminary toxicity test where the onset of precipitate was considered to effectively reduce exposure of the test item to the cells. It was therefore considered that, based on the modest decreases in RTG and %RSG values observed at 51 µg/mL in the absence of metabolic activation, and also the precipitate observations, maximum exposure of the test item to the cells had been achieved at 51 to 68 µg/mL. The test item was, therefore, considered to have been adequately tested within the parameters stated in the OECD 476 (1997) test guideline. Acceptable levels of toxicity were seen with both positive control substances (Tables 3 and 6).

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 3 and 6).

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). A precipitate of the test item was observed at 51 µg/mL in the absence of metabolic activation, and became greasy / oily in appearance at 68 µg/mL in both of the exposure groups.

The numbers of small and large colonies and their analysis are presented in attached Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in attached Tables 8 to 13.

As was seen previously, there was evidence of significant toxicity in the absence of metabolic activation, as indicated by the %RSG and RTG values (Table 9). On this occasion there were also some very modest reductions observed in the presence of metabolic activation (Table 12). There was no evidence of any significant reductions in viability (%V) in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred in either of the exposure groups (Tables 9 and 12). Based on the RTG and %RSG values observed, and also the precipitate observations, it was considered that maximum exposure of the test item to the cells had been achieved at 51 to 68 µg/mL (Tables 9 and 12). Acceptable levels of toxicity were seen with both positive control substances (Tables 9 and 12).

The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had a significant effect on the toxicity of the test item. It should also be noted that the lowering of the S9 concentration to 1% in this second experiment resulted in slightly higher levels of toxicity being observed when compared to 4-hour exposure groups in the presence of 2% metabolic activation in the Preliminary Toxicity Test and Experiment 1.

The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional (Tables 9 and 12).

The test item did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 9 and 12). Precipitate of test item was observed at and above 51 µg/mL in both the absence and presence of metabolic activation, and appeared greasy / oily at 68 µg/mL in the presence of metabolic activation.

The numbers of small and large colonies and their analysis are presented in attached Tables 10 and 13.
Remarks on result:
other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Remarks:
Migrated from field 'Test system'.

Please see attached "Tables 1 to 13"

Due to the nature and quantity of the tables it was not possible to insert them in this section.

Conclusions:
Interpretation of results: negative

The test item, Farnesane, did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.
Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item, Farnesane, on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.476 "In Vitro Mammalian Cell Gene Mutation Tests" adopted 21 July 1997, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

 

Methods…….

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels, in duplicate, together with vehicle (acetone) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test item at six dose levels using a 4-hour exposure group in the presence of metabolic activation (1% S9) and a 24-hour exposure group in the absence of metabolic activation.

 

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. Formulation analysis was performed on the highest dose concentration of Experiment 1. 

Results……..

The maximum dose level used was limited by the onset of greasy / oily precipitate effectively reducing exposure of the test item to the cells. Overall, precipitate of the test item was observed at and above 51 µg/mL in the Mutagenicity Test. The vehicle controls (acetone) had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control treatment induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

 

The test item did not induce any toxicologically significant dose-related (linear-trend) increases in the mutant frequency at any of the dose levels, either with or without metabolic activation, in either the first or the second experiment.

Conclusion

The test item, Farnesane, did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic under the conditions of the test.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Farnesane was not mutagenic in an Ames Assay; Not clastogenic in cultured human peripheral blood lymphocytes in both the absence and presence of S-9 when tested up to 2000 µg/mL in a Guideline 473 chromosome aberration study; and did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells in an in vitro mammalian gene mutation assay.

Based on the weight of evidence available from in vitro genetic toxicity assays, Farnesane does not appear to be mutagenic and is therefore not classified under CLP for mutagenicity.