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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in-vitro genotoxicity studies are available for farnesene - Ames Assay, chromosomal aberration study and a mouse lymphoma assay. All three studies were carried out in accordance with OECD guidelines. None of the studies showed any evidence of genotoxic activity. Hence Farnesene is not considered to be genotoxic and does not need to be classified according to the CLP Regulation (EC) N° (1272-2008).

Link to relevant study records
Reference
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:
01/09/09-03/09/09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
equivalent or similar to guideline
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: E. coli WP2 uvrA, and S. typhimurium strains TA-97a, TA-1535, TA-98, and TA-100
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:
Five concentrations (0.05, 0.16, 0.5, 1.6 and 5 μl/plate, 3 plates per dose) of the test article were tested in each of five bacterial tester strains (Escherichia coli (E. coli) WP2 uvrA, and S. typhimurium strains TA97a, TA1535, TA98, and TA100) both with and without metabolic activation (= s plates)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test article was freely soluble at a concentration of 50 μl/ml in DMSO
Untreated negative controls:
no
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 ±1ºC with agitation. The cultures were used after they reached 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 ±1ºC for 48-72 hours. Plates that were not scored immediately following the incubation period but 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 (0.05, 0.16, 0.5, 1.6 and 5 μl/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.

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.
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
A dose-related decrease of revertant frequencies found in most concentrations in TA97a raised a concern that test concentrations were cytotoxic. In an independent repeat assay, no obvious cytotoxicity found based on the bacterial background lawn check
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
A dose-related decrease of revertant frequencies found in most concentrations in TA100 raised a concern that test concentrations were cytotoxic. In an independent repeat assay, no obvious cytotoxicity found based on the bacterial background lawn check
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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
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 (migrated information): negative

Under test conditions for both main and independent repeat assays, the test article, trans-ß-farnesene, CAS#18794-84-8, was negative for mutagenicity in the Bacterial Reverse Mutation Assay (Ames Assay).
Executive summary:

Method Synopsis: Prior to the cytotoxic screen, solubility of the test article was checked in Dimethyl sulfoxide (DMSO). The test article was freely soluble at a concentration of 50 μl/ml in DMSO. Therefore, DMSO was chosen as the vehicle in the main assay. A cytotoxicity screen was conducted in the TA100

tester strain using eight concentrations (0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1 and 5 μl) of the test article, two plates per dose, on the bacterial tester strain Salmonella typhimurium (S. typh.)TA100. The test article was combined with the bacteria and top agar in the presence and absence of a metabolic activation buffer containing S9 and overlaid onto minimal glucose agar plates. A DMSO vehicle control was run concurrently, with S9.

Based on the cytotoxicity results, five concentrations (0.05, 0.16, 0.5, 1.6 and 5 μl/plate, 2 plates per dose) of the test article were tested in each of five bacterial tester strains (Escherichia coli (E. coli) WP2 uvrA, and S. typhimurium strains TA97a, TA1535, TA98, and TA100). 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 ±1ºC 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.

Due to negative results in the main assay and cytotoxicity found in the entire dose range in strains TA97a, and TA100, an independent repeat assay was conducted in all tester strains. Test article concentrations of 0.05, 0.16, 0.5, 1.6 and 5 μl/plate for strains WP2, TA98, TA1535 (all both with and without S9), and TA97A and TA100 (both without S9 only). Concentrations of 0.005, 0.016, 0.05, 0.16, 0.5 μl/plate were used for strains TA97A and TA100 (both with S9 only).

Results summary: In the screen, bacterial background lawn checks and colony counts indicated that the test article at 5 and 1 μl/plate was slightly cytotoxic to TA100. In the main assay, slight cytotoxicity of the test article, based on the bacterial background lawn checks, was observed in strains TA97a (1.6 μl/plate with S9 and 5 μl/plate without S9), TA100 (5 μl/plate with and without S9), and TA 1535 (5 μl/plate with S9 only). No positive response or dose-related increased response of the test article in any strain was found in the assay.

However, a dose-related decrease of revertant frequencies found in most concentrations in strains TA97a and TA100, with S9, raised a concern that these concentrations were cytotoxic. In the independent repeat assay, no obvious cytotoxicity, based on the bacterial background lawn check, was observed in any of the strains. As in the main assay, no positive response or concentration-related increase response in any strain was found.

Conclusion: Under test conditions for both main and independent repeat assays, the test article, trans-ß-farnesene, CAS#18794-84-8, was negative for mutagenicity in the Bacterial Reverse Mutation Assay (Ames Assay).

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

Additional information

Additional information from genetic toxicity in vitro:

Three guideline genotoxicity studies are available for Farnesene. Details are summarised as follows:

Ames study: In a screening study, bacterial background lawn checks and colony counts indicated that the test article at 5 and 1 μl/plate was slightly cytotoxic to TA100. In the main assay, slight cytotoxicity of the test article, based on the bacterial background lawn checks, was observed in strains TA97a (1.6 μl/plate with S9 and 5 μl/plate without S9), TA100 (5 μl/plate with and without S9), and TA 1535 (5 μl/plate with S9 only).

No positive response or dose-related increased response of the test article in any strain was found in the assay. However, a dose-related decrease of revertant frequencies found in most concentrations in strains TA97a and TA100, with S9, raised a concern that these concentrations were cytotoxic. In an independent repeat assay, no obvious cytotoxicity, based on the bacterial background lawn check, was observed in any of the strains.

As in the main assay, no positive response or concentration-related increase response in any strain was found it is concluded that farnesene was not mutagenic.

Lymphocyte Chromosomal Aberration Assay:

In a study conducted to OECD guideline 473, human lymphocytes were exposed to farnesene dissolved in DMSO, at concentrations of 6.25, 12.5, 25, 31.25, 50 and 75 μg/mL in the presence of S-9 mix. Incubations in the absence of S-9 mix were carried out at 3.125, 6.25, 12,5,31.25 and 50 μg/mL. Positive control materials tested (mitomycin C and cyclophosphamide) gave acceptable responses, showing a valid test system

Positive controls (mitomycin C without S9, cyclophosphamide with S9) induced the appropriate response. Vehicle controls were also valid. Farnesene was moderately toxic but did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included dose level that approached the 50% mitotic inhibition limit. It was concluded that farnesene was not clastogenic to human lymphocytes in-vitro.

Mouse Lymphoma Assay

The study was conducted using mouse lymphoma L5178Y cells and according to OECD method 476. 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 farnesene at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9 final concentration). In Experiment 2, the cells were treated with farnesene at eight dose levels using a 4‑hour exposure group in the presence of metabolic activation (2% S9 final concentration) and a 24‑hour exposure group in the absence of metabolic activation. The dose range of test item was selected following the results of a preliminary toxicity test and for Experiment 1 was 0.25 to 12 µg/ml in the absence of metabolic activation, and 1 to 64 µg/ml in the presence of metabolic activation. In Experiment 2 the dose range was 0.5 to 20 µg/ml in the absence of metabolic activation, and 2 to 48 µg/ml in the presence of metabolic activation.

The maximum dose levels used in the assay were limited by test item-induced toxicity. Precipitate of the test item was not observed at any of the dose levels in the Mutagenicity Test. The vehicle (solvent) controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

Farnesene did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or second experiment. It was concluded that Farnesene was not mutagenic to L5178Y cells under the conditions of the test.



Justification for selection of genetic toxicity endpoint
One of 3, Klimisch 1, guideline studies carried out on Farnesene. All showed negative results

Justification for classification or non-classification

Farnesene showed no evidence of genotoxicity in three in-vitro assay systems and does not need to be classified according to the CLP Regulation (EC) N° (1272-2008).