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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non-mutagenic (OECD 471 and OECD 476) and non-clastogenic (OECD 473)

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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:
05 December 2011 to 24 January 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection 2011-07-19 to 2011-07-21; Date of signature 2011-08-31
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.
- Properly maintained: yes - The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly.
- Periodically "cleansed" against high spontaneous background: yes - Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal enzyme fraction
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 7.19, 14.38, 28.75, 57.5, 115, 230, 460, 920 and 1840 µg/mL

Experiment 1 (without S-9 mix): 0, 1.25, 2.5, 5, 10, 20, 40, 50 and 60 µg/mL
Experiment 1 (with S-9 mix): 0, 5, 10, 20, 40, 60, 80, 100 and 120 µg/mL

Experiment 2 (without S-9 mix): 0, 2.5, 5, 10, 20, 40, 50, 60 and 80 µg/mL
Experiment 2 (with S-9 mix): 0, 10, 20, 40, 60, 80, 100, 120 and 140 µg/mL
Vehicle / solvent:
The test material was formulated in dimethyl sulphoxide (DMSO).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Experiment 1: 400 µg/mL; Experiment 2: 150 µg/mL; without metabolic activation
Positive control substance:
cyclophosphamide
Remarks:
2 µg/mL; with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

PRELIMINARY TOXICITY TEST
A preliminary toxicity test was performed on cell cultures at 5E+05 cells/mL using a 4 hour exposure time both with and without metabolic activation and at 1.5E+05 cells/mL using a 24 hour exposure period without S9. The dose range used in the preliminary toxicity test was 7.19 to 1840 µg/mL for all 3 of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2E+05 cells/mL.
The cultures were incubated at 37°C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2E+05 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post treatment toxicity and a comparison of each treatment SG value to the concurrent vehicle control performed to give a % Relative Suspension Growth (%RSG) value.
Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments.

MUTAGENICITY TEST: EXPERIMENT 1
Cells were counted and processed to give 1E+06 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. The treatments were performed in duplicate both with and without metabolic activation at 8 dose levels of the test item (1.25 to 60 µg/mL in the absence of metabolic activation and 5 to 120 µg/mL in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9-mix (if required), 0.2 mL of the treatment dilutions, 0.2 mL for the positive control and sufficient R0 medium to bring the total volume to 20 mL.
The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking.

MUTAGENICITY TEST: EXPERIMENT 2
Cells were counted and processed to give 1E+06 cells/mL in 10 mL duplicate cultures in R10 medium for the 4 hour treatment with metabolic activation cultures. In the absence of metabolic activation the exposure period was extended to 24 hours; therefore, 0.3E+06 cells/mL in 10 mL duplicate cultures were established in 25 cm2 tissue culture flasks. To each culture 2 mL of S9-mix was added (if required), 0.2 mL of the treatment dilutions, 0.2 mL for the positive control and sufficient R0 medium to give a final volume of 20 mL (R10 is used for the 24 hour exposure group). The dose range of the test item was 2.5 to 80 µg/mL in the absence of metabolic activation and 10 to 140 µg/mL in the presence of metabolic activation.
The treatment vessels were incubated at 37 °C with continuous shaking for 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

MICROSOMAL ENZYME FRACTION
PB/βNF S9 microsomal fraction was prepared from the livers of male Sprague-Dawley rats weighing approximately 250 g. These had each received, orally, 3 consecutive daily doses of Phenobarbitone/β-Naphthoflavone (80/100 mg/kg/day) prior to S9 preparation on day 4. Aliquots of supernatant were frozen and stored at approximately -196 °C.
The S9-mix was prepared by mixing S9, 33 mM KCl, 8 mM MgCl2, 5 mM glucose-6-phosphate and 5 mM NADP in R0.
20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the preliminary toxicity test and of Experiment 1. In Experiment 2, 0% S9-mix (i.e. 1% final concentration of S9) was added.

DURATION
- Exposure duration: 4-hour exposure with and without metabolic activation in Experiment 1 and with metabolic activation in Experiment 2 and 24-hour exposure without metabolic activation in Experiment 2.
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): On Day 2 of the experiment, the cells were counted, diluted to 10E+04 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

STAIN: To assist the scoring of the TFT mutant colonies 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours.

SELECTION AGENT (mutation assays): 5 trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Each treatment was performed in duplicate (A + B), both with and without metabolic activation (S9-mix).

MEAUREMENT OF SURVIVAL, VIABILITY AND MUTANT FREQUENCY
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2E+05 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of 2 days by counting and diluting to 2E+05 cells/mL. On Day 2 of the experiment, the cells were counted, diluted to 10E+04 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
The daily cell counts were used to obtain a %RSG value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control and when combined with the %V data a Relative Total Growth (RTG) value.

PLATE SCORING
Microtitre plates were scored after 10 to 14 days incubation at 37 °C with 5% CO2 in air. To assist the scoring of the TFT mutant colonies 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The normal range for mutant frequency per survivor is 50-200 x 10E-06 for the TK+/- locus in L5178Y cells. Vehicle control results should ideally be within this range.
Positive control chemicals should induce at least 3 to 5 fold increases in mutant frequency greater than the corresponding vehicle control.
For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST
In all 3 of the exposure groups there was evidence of marked dose-related reductions in the %RSG of cells treated with the test item when compared to the concurrent vehicle controls. The steep nature of the toxicity curve was taken to indicate that achieving optimum toxicity would be difficult. A greasy/oily precipitate was observed at and above 230 µg/mL at the end of the exposure period in all 3 of the exposure groups. In addition, an increase in intensity was associated with an increase in dose concentration. Based on %RSG values observed, the maximum dose levels in the subsequent mutagenicity test were limited by test item induced toxicity.

MUTAGENICITIY TEST - EXPERIMENTS 1 AND 2:
Based on the %RSG and RTG values observed, it was considered that optimum levels of toxicity had been achieved in the presence of metabolic activation and very near optimum levels of toxicity in the absence of metabolic activation. Acceptable levels of toxicity were seen with both positive control substances.
The 24 hour exposure in Experiment 2 without metabolic activation demonstrated that the extended time point had no marked effect on the toxicity of the test item.
Neither of the vehicle control mutant frequency values were outside the acceptable range. Both of the positive controls produced marked increases in the mutant frequency per viable cell.
The test item did not induce any statistically significant or dose related increases in the mutant frequency x10E-06 per viable cell in either the absence or presence of metabolic activation.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate of test item was not observed at any of the dose levels in either Experiment.
Conclusions:
The test item, 9-decenoic acid, methyl ester (9DAME) 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:
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:
19 October 2011 - 08 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection 2011-07-19 to 2011-07-21; Date of signature 2011-08-31
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 microsomal enzyme fraction
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate
Mutation test - Experiment 1:
- Salmonella strains TA1535 and TA1537 (without S-9 mix only): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- All other bacterial strains (with and without S-9 mix): 50, 150, 500, 1500 and 5000 μg/plate
Mutation test - Experiment 2:
- All bacterial strains (without S-9 mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate
- All bacterial strains (with S-9 mix): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration. Dimethyl sulphoxide was therefore selected as the vehicle.
Untreated negative controls:
yes
Remarks:
Concurrent untreated controls (spontaneous mutation rates)
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
2 µg/plate for WP2uvrA, 3 µg/plate for TA100 and 5 µg/plate for TA1535 without metabolic activation
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537 without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 µg/plate for TA98 without metabolic activation
Positive control substance:
other: 2-aminoanthracene
Remarks:
10 µg/plate for WP2uvrA, 1 µg/plate for TA100, 2 µg/plate for TA1535 and 2 µg/plate for TA1537 with metabolic activation
Positive control substance:
benzo(a)pyrene
Remarks:
5 µg/plate for TA98 with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and preincubation for Experiment 2.

PRELIMINARY TOXICITY TEST
- The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate and a vehicle control.
- The test was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA), 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of test item formulation and 0.5 mL of S-9 mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate).
- After approximately 48 hours incubation at 37 °C the plates were assessed for numbers of revertant colonies and examined for effects on the growth of the bacterial background lawn. After approximately 48 hours incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Test - Experiment 1:
Up to 7 concentrations of the test item were assayed in triplicate against each tester strain, using the direct plate incorporation method. Dose ranges were allocated as follows:
- Salmonella strains TA1535 and TA1537 (without S-9 mix only): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- All other bacterial strains (with and without S-9 mix): 50, 150, 500, 1500 and 5000 μg/plate
Additional dose levels and an expanded dose range were selected (where applicable) in order to achieve both four non-toxic dose levels and the toxic limit of the test item.
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation Test - Experiment 2:
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was amended, following the results of Experiment 1 and the change in test methodology, and was as follows:
- All bacterial strains (without S-9 mix): 0.15, 0.5, 1.5, 5, 15, 50 and 150 µg/plate
- All bacterial strains (with S-9 mix): 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Additional dose levels and an expanded dose range were selected for each bacterial strain in order to achieve both four non-toxic dose levels and the toxic limit of the test item.
The test material formulations and vehicle control were dosed using the pre-incubation method as follows:
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix. The positive and untreated controls were dosed using the standard plate incorporation method described above.
All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

DURATION
- Preincubation period: 20 minutes (Experiment 2)
- Exposure duration: 48 h (Experiment 1)

NUMBER OF REPLICATIONS: Plates were prepared in triplicate

NUMBER OF CELLS EVALUATED: All tester strain cultures should be in the range of 0.9 to 9 x 10^09 bacteria per mL.

MICROSOMAL ENZYME FRACTION
The S9 microsomal fraction was prepared from rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenising the liver in a 0.15 M KCl solution followed by centrifugation at 9000g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of supernatant were frozen and stored at approximately -196 °C.

S9-MIX
The S9-mix was prepared immediately before use using sterilised co-factors (see below) and maintained on ice for the duration of the test.
S9: 5.0 mL
1.65M KCl/0.4 M MgCl2: 1.0 mL
0.1 M glucose-6-phosphate: 2.5 mL
0.1 M NADP: 2.0 mL
0.2 M sodium phosphate buffer (pH 7.4): 25.0 mL
Sterile distilled water: 14.5 mL
Evaluation criteria:
There were several criteria for determining a positive result:

1). A dose-related increase in mutant frequency over the dose range tested.
2). A reproducible increase at one or more concentrations.
3). Biological relevance against in-house historical control ranges.
4). Statistical analysis of data as determined by UKEMS.
5). Fold increase greater than 2 times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item was considered non-mutagenic (negative) in the test system if the above criteria were not met.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S-9 mix.

PRELIMINARY TOXICITY TEST
The test item was non-toxic to the strains of bacteria used (TA100 and WP2uvrA). The test formulation and S9-mix used in this experiment were both shown to be sterile.

MUTATION TEST:
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.

In Experiment 1 (plate incorporation) the test item caused a visible reduction in the growth of the bacterial background lawns of Salmonella strains TA1535 and TA1537 from 1500 µg/plate in the absence of S-9 mix only. No toxicity was noted for TA100, TA98 and WP2uvrA (absence of S-9 mix) or any strain dosed in the presence of S-9 mix. In Experiment 2 (following pre-incubation methodology) weakened bacterial background lawns were noted to all of the bacterial strains except WP2uvrA (presence of S-9 mix), initially from 50 µg/plate in the absence of S-9 mix and 1500 µg/plate in the presence of S-9 mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied between strain type, exposures with or without S-9 mix and experimental methodology.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Conclusions:
The test item, 9-decenoic acid, methyl ester (9DAME) was considered to be non-mutagenic under the conditions of this test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 October 2011 to 24 January 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection 2011-07-19 to 2011-07-21; Date of signature 2011-08-31
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 16 hours under typical experimental exposure conditions.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 7.19, 14.38, 28.75, 57.5, 115, 230, 460, 920 and 1840 µg/mL
Experiment 1: 0, 15, 30, 60, 75, 90 and 120 µg/mL
Experiment 2: 0, 7.5, 15, 30, 60, 90, 120 and 180 µg/mL
Vehicle / solvent:
The test item was accurately weighed, dissolved in dimethyl sulphoxide (DMSO) and serial dilutions prepared.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9: 0.4 µg/mL and 0.2 µg/mL for 4(20) hour and 24 h cultures, respectively
Positive control substance:
cyclophosphamide
Remarks:
With S9: 5 µg/mL for Experiment 1 and 2
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

MICROSOMAL ENZYME FRACTION
The S9 microsomal fraction was prepared from rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenising the liver in a 0.15 M KCl solution followed by centrifugation at 9000g. The protein content of the resultant supernatant was adjusted to 20 mg/mL using 0.15 M KCl. Aliquots of supernatant were frozen and stored at approximately -196 °C.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (10 to 20% (v/v)), KCl (33 mM), MgCl2 (8 mM), sodium orthopohosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM) and NADP (5 mM). The final concentration of S9, when dosed at a 10% volume of S9 mix into culture media was 2% in the Preliminary Toxicity Test and Experiment 1 and 1% in Experiment 2.

METHOD WITH METABOLIC ACTIVATION
After approximately 48 hours incubation at 37 °C, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved and replaced with the required volume of MEM and 0.1 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1 mL of 20% S9 mix (i.e. 2% final concentration of S9 in standard cofactors) was added to the cultures of the Preliminary Toxicity Test and of Experiment 1. In Experiment 2, 1 mL of 10% S9 mix (i.e. 1% final concentration of S9 in standard cofactors) was added. All cultures were then returned to the incubator. The nominal final volume of each culture was 10 mL.
After 4 hours at 37 °C, 5% CO2 in humidified air the cultures were centrifuged, the treatment medium removed and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at 37 °C in 5% CO2 in humidified air.

METHOD WITHOUT METABOLIC ACTIVATION
In Experiment 1, after approximately 48 hours incubation at 37 °C, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM and dosed with 0.1 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL.
After 4 hours at 37 °C, 5% CO2 in humidified air the cultures were centrifuged, the treatment medium removed and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours.
In Experiment 2, in the absence of metabolic activation, the exposure was continuous for 24 hours. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at 37 °C, 5% CO2 in humidified air for 24 hours.
The Preliminary Toxicity Test was performed using the methodology of Experiment 1 and Experiment 2 in the absence of metabolic activation except that positive controls were not included.

PRELIMINARY TOXICITY TEST
A preliminary toxicity test was performed on cell cultures using a 4 hour exposure time with and without metabolic activation followed by a 20 hour recovery period and a continuous exposure of 24 hours without metabolic activation. The dose range of test item used was 7.19 to 1840 µg/mL. Parallel flasks, containing culture medium without whole blood were established for the three exposure conditions so that test item precipitate observations could be made (recorded at the beginning and end of the exposure periods).
Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

EXPERIMENT 1
1). 4 hour exposure to the test item without S9-mix followed by 20 hour culture in treatment free media prior to cell harvest. The dose range of the test item used was 15 to 120 µg/mL.
2). 4 hour exposure to the test item with S9-mix followed by 20 hour culture in treatment free media prior to cell harvest. The dose range of the test item used was 15 to 120 µg/mL.

EXPERIMENT 2
1). 24 hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of the test item used was 7.5 to 180 µg/mL.
2). 4 hour exposure to the test item with S9-mix followed by 20 hour culture in treatment free media prior to cell harvest. The dose range of the test item used was 7.5 to 180 µg/mL.

DURATION
- Preincubation period: 48 hours
- Exposure duration: Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9; 4 hrs with S9
- Expression time (cells in growth medium): 20 hrs for 4 hr exposure
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Demecolcine

STAIN (for cytogenetic assays): When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.

NUMBER OF REPLICATIONS: duplicate

SCORING OF CHROMOSOME DAMAGE
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index. A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 57.5 µg/mL in the 4(20) hour exposure in the absence of metabolic activation (S9). In the presence of S9 metaphase cells were present in the maximum dose of 1840 µg/mL, however, there was a reduction in cells and metaphases noted in the qualitative assessment of the slides at and above 230 µg/mL, indicative of toxicity. The vehicle control value in the 4(20) hour exposure group in the presence of S9 was considered to be low resulting in the mitotic index values being artificially raised and therefore not giving a true indication of toxicity. The maximum dose with metaphases present in the 24 hour continuous exposure was 115 µg/mL.

EXPERIMENT 1
The mitotic index data confirm the qualitative observations in that a dose related inhibition of mitotic index was observed in the presence of S9 and 59% mitotic inhibition was achieved at 75 µg/mL. In the absence of S9, 40% mitotic inhibition was achieved at 90 µg/mL which was just before the onset of greasy/oily precipitate.
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations.
The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.
The test item did not induce any statistically significant increases in the numbers of polyploid cells at any dose level in either of the exposure groups.

EXPERIMENT 2
The mitotic index data confirm the qualitative observations in that a dose related inhibition of mitotic index was observed and that 54% mitotic inhibition was achieved at 120 µg/mL in the absence of S9. The toxicity seen in the 24 hour exposure group was comparable with that seen in the Preliminary Toxicity Test. In the presence of S9, 52% and 40% mitotic inhibition were achieved at 120 and 180 µg/mL, respectively. The difference in toxicity between Experiment 1 and 2 in the 4(20) hour exposure group in the presence of S9 is considered to be due to the reduction in S9 concentration.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations.
The test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation.
The test item did not induce any statistically significant increases in the numbers of polyploid cells at any dose level in either of the exposure groups.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Preliminary Toxicity Test: A greasy/oily precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 115 µg/mL in the 4(20) hour exposure groups and at and above 230 µg/mL in the 24 hour continuous exposure group. A cloudy precipitate was also observed in the presence of S9 at and above 920 µg/mL. A cloudy precipitate was observed along with modest greasy/oily precipitate when cultures were initially dosed at and above 230 µg/mL and this was lost or reduced during the incubation of the cultures. This transitional phase as the test item aggregated to create greasy/oily precipitate at and above 115 µg/mL in the 4(20) hour cultures in the 15 mL tubes could be instrumental in the varying toxicity observed in the two main experiments.
Experiments 1 and 2: No precipitate of the test item was observed at the end of exposure, in either exposure group.
- Other confounding effects: Haemolysis was observed in the Preliminary Toxicity Test in the blood cultures at the end of the exposure at and above 14.38 µg/mL in the 4(20) hour exposure groups and at and above 57.5 µg/mL in the 24 hour exposure group. However, the observation of haemolysis has no effect on the endpoint of this test. Haemolysis was observed at and above 15 µg/mL in the absence of S9 and 30 µg/mL in the presence of S9 for Experiments 1 and 2. In Experiment 2 there was also a reduction in the size of the cell pellet noted at the end of exposure in both exposure groups at and above 30 µg/mL which is indicative of the increasing toxicity above this dose level.
Conclusions:
The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Additional information from genetic toxicity in vitro:

In vitro gene mutation study in bacteria:

In a reliable GLP OECD guideline 471 study (Harlan Laboratories Ltd, 2012), 9-decenoic acid, methyl ester (9-DAME) was tested in a bacterial reverse mutation assay in Salmonella typhimurium (strains TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA with and without metabolic activation (S9). The concentrations tested were between 5 and 5,000μg/plate in the first experiment and between 0.15 and 5000 µg/plate in Experiment 2. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. The test item, 9-decenoic acid, methyl ester (9-DAME) was therefore considered to be non-mutagenic.

 

In vitro chromosome aberration study in mammalian cells:

In a reliable GLP OECD guideline 473 study (Harlan Laboratories Ltd, 2012), 9-decenoic acid, methyl ester (9-DAME) was tested for its ability to induce structural chromosomal aberrations in cultured mammalian cells (human lymphocytes) in the presence and absence of metabolic activation. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition. 9-decenoic acid, methyl ester was therefore considered to be non-clastogenic to human lymphocytes in vitro.

 

In vitro gene mutation study in mammalian cells:

In a reliable GLP OECD guideline 476 study (Harlan Laboratories Ltd, 2012), 9-decenoic acid, methyl ester (9-DAME) was tested for its ability to induce mutations in mouse lymphoma L5178Y cells in the presence and absence of metabolic activation. The test item 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. 9-decenoic acid, methyl ester (9-DAME) was therefore considered to be non mutagenic to L5178Y cells under the conditions of the test.

 

Justification for classification or non-classification

The results of an in vitro gene mutation study in bacteria, an in vitro chromosome aberration study in mammalian cells and an in vitro gene mutation study in mammalian cells were all negative. Therefore, it is concluded that 9-decenoic acid, methyl ester (9-DAME) is not genotoxic and does not warrant classification for mutagenicity according to CLP Regulation EC 1272/2008.