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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 27 February 2012 to 2 April 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD Guideline 476 without any deviations. γ-Nonalactone, as a linear saturated 4-hydroxycarboxylic acid derived-lactones, is considered adequate for read-across purpose (see §"Toxicokinetics").
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):Gamma-Nonalactone
- Substance type: lactone
- Physical state: Transparent clear to pale yellow liquid
- Analytical purity:98.64%
- Impurities (identity and concentrations):no data
- Lot/batch No.:312125.00549
- Expiration date of the lot/batch:19 October 2012
- Stability under test conditions: stable (sponsor information)
- Storage condition of test material: Room temperature, protected from light

Method

Target gene:
The L5178Y mouse lymphoma (3.7.2c) cells are heterozygous at the thymidine kinase locus, TK +/-
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: R10p medium was used for cell culture unless otherwise specified. R20p medium was used for the cloning efficiency plating. This was prepared by mixing equal volumes of R10p and R30p.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:
with and without
Metabolic activation system:
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6 benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at ca -80°C. Lot No.: 2882 (Date of preparation
Test concentrations with justification for top dose:
Preliminary toxicity test: -S9 mix and +S9 mix
(3 hours and 24 hours) 3.1, 6.1, 12.2, 24.4, 48.8, 97.6, 195.3, 390.5, 781 and 1562 µg/mL

Mutation tests:
-S9 mix (3 hours) 200, 300, 400, 450, 500, 550, 600, 650, 700, 750 and 800 µg/mL
+S9 mix (3 hours) 200, 300, 400, 600, 800, 1000, 1200, 1400 and 1562 µg/mL

+S9 mix additional (3 hours) 200, 400, 800, 1000, 1050, 1100, 1150 and 1200 µg/mL

-S9 mix (24 hours) 100, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700 and 800 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Gamma-Nonalactone was dissolved and diluted in acetone (analytical grade), shortly before dosing. The final concentration of acetone added to the cultures was 1% v/v.
- Justification for choice of solvent/vehicle: Prior to commencing testing, Gamma-Nonalactone was confirmed to be soluble at 156.2 mg/mL in acetone. A solution of 1562 mg/mL, dosed at 1% in medium, showed no precipitate in the culture medium
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
In the absence of S9 mix
Positive control substance:
methylmethanesulfonate
Remarks:
Solvent: DMSO / Exposure concentration:10 µg/mL (3 hour exposure) and 5 µg/mL (24 hour exposure)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
In the presence of S9 mix
Positive control substance:
benzo(a)pyrene
Remarks:
Solvent: DMSO / Exposure concentration:10 µg/mL (3 hour exposure)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 hours or 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): the mutant 96-well plates had been incubated 10 to 14 days
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): Selective medium consisted of R10p containing 4 µg/mL trifluorothymidine (TFT).

NUMBER OF REPLICATIONS: Duplicate cultures were prepared throughout for each concentration of test substance and positive control. Quadruplicate cultures were prepared for vehicle controls.

NUMBER OF CELLS EVALUATED: Cloning efficiency was assessed by plating 1.6 cells/well in R20p medium, two plates being prepared per culture. Mutant potential was assessed by plating 2 x 103 cells/well in selective medium, two plates being prepared per culture.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (CE); relative suspention growth (RSG); other: mutant frequency (MF)

OTHER EXAMINATIONS:
- Other: The colony size distribution in the vehicle and positive controls was examined to ensure that there was an adequate recovery of small colony mutants.
Evaluation criteria:
On completion of each main mutagenicity test, data were examined for cell growth parameters, cytotoxicity, plating efficiencies, spontaneous and positive control MF, and percent small colonies in positive control cultures.
The criteria used to assess whether an assay was valid are based on the recommendations of the Plymouth and Aberdeen papers (Robinson et al., 1989; Moore et al., 2000; Moore et al., 2002; Moore et al., 2003; Moore et al., 2006 and Moore et al., 2007).

Acceptance criteria for test substance:
The highest concentration tested was one that allowed the maximum exposure up to 5000 µg/mL or 10 mM for freely soluble compounds, or the limit of toxicity (ie. relative total growth reduced to approximately 10 to 20% of the concurrent vehicle control) or the limit of solubility. For a toxic substance, at least 4 analysable concentrations should have been achieved which ideally spanned the toxicity range of 100 to 10% RTG.

Acceptance criteria for vehicle controls:
The mean vehicle control value for mutant frequency was between 50 to 170 x 10-6.
The mean cloning efficiency was between 65 to 120%.
The mean suspension growth was between 8 to 32 on Day 2 following 3 hour treatments and between 32 to 180 on Day 2 following a 24 hour treatment.
Obvious outliers were excluded. However, there were at least 2 vehicle control cultures remaining.

Acceptance criteria for positive controls:
Positive controls showed an absolute increase in mean total MF above the mean concurrent vehicle control MF of at least 300 x 10-6. At least 40% of this was due to the number of small mutant colonies.
Mean RTG’s for the positive controls were greater than 10%.
There was an absence of confounding technical problems such as contamination, excessive numbers of outliers and excessive toxicity.
There was not excessive heterogeneity between replicate cultures.
Statistics:
The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1, which follows the methods described by Robinson et al. (1989) using a one-sided F-test, where p<0.001. Statistics were only reported if the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor was exceeded, and this was accompanied by a significant positive linear trend.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
up to Gamma-Nonalactone cytotoxic concentrations following 3-hour and 24-hour exposure.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
No precipitate (observed by eye at the end of treatment) was observed at any concentration tested in the absence and presence of S9 mix following any exposure.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality:no
- Evaporation from medium:no
- Water solubility:no
- Precipitation:no
- Other confounding effects:no

RANGE-FINDING/SCREENING STUDIES: no

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: No precipitate (observed by eye at the end of treatment) was observed at any concentration tested in the absence and presence of S9 mix following a 3 hour exposure. Exposure to Gamma-Nonalactone at concentrations from 3.1 to 1562 µg/mL in the absence and presence of S9 mix (3 hour exposure) resulted in relative suspension growth (RSG) values between 132 and 0% and between 109 and 2% respectively. (see Table 1 in attached document)
Following a continuous exposure for 24 hours, no precipitation (assessed by eye at the end of treatment) was observed at any concentration tested. Exposure to concentrations from 3.1 to 1562 µg/mL resulted in RSG values between 104 and 0%. (see Table 1 in attached document)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

1.1 Main mutation test - 3 hour treatment in the absence of S9 mix (see Table 2 and Table 3 in attached document)

Cultures were exposed to Gamma-Nonalactone at concentrations from 200 to 800 mg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Gamma-Nonalactone at concentrations from 200 to 550 mg/mL were assessed for determination of mutation frequency. Relative total growth (RTG) values from 125 to 23% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum (213 x 10-6), of the mean concurrent vehicle control mutant frequency (87 x 10-6) and the Global Evaluation Factor (GEF) of 126 x 10-6, within acceptable levels of toxicity.

Although this assay did not reach the required 10-20% RTG it is considered valid in accordance withMoore et al. (2002)which states that a chemical can be considered nonmutagenic when there is no culture showing an RTG value between 10-20% if there is no evidence of mutagenicity (e.g., no dose response or mutant frequencies above those seen in the historical background ranges) in a series of data points within 100% to 20% RTG and there is at least one negative data point between 20% and 25% RTG.

The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

1.2  Main mutation test - 3 hour treatment in the presence of S9 mix (see Table 4 in attached document)

The results are shown inTable4.

Cultures were exposed to Gamma-Nonalactone at concentrations from 200 to 1562 mg/mL. No precipitate was observed by eye at the end of treatment. RSG values from 90 to 2% were obtained relative to the vehicle control. As an appropriate toxicity profile was not achieved an additional test was performed.

1.3 Additional main mutation test - 3 hour treatment in the presence of S9 mix (see Table 5 and Table 6 in attached document)

Cultures were exposed to Gamma-Nonalactone at concentrations from 200 to 1200 mg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Gamma-Nonalactone at concentrations from 200 to 1050 mg/mL were assessed for determination of mutation frequency. RTG values from 110 to 11% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum (214 x 10-6) of the mean concurrent vehicle control mutant frequency (88 x 10-6) and the GEF (126 x 10-6), within acceptable levels of toxicity.

The positive control, benzo[a]pyrene,induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

The results obtained in response to the exposure of cultures to Gamma-Nonalactone in the presence of S9 mix did not demonstrate mutagenic potential. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF, within acceptable levels of toxicity. All mean mutant frequencies of the test concentrations were within the acceptable vehicle control values and there were no clear increases in the mean mutant frequencies of any test concentration assessed that were associated with a linear trend (P>0.05). Therefore it was considered not to be beneficial to perform a direct repeat of the assay.

1.4    Main mutation test - 24 hour treatment in the absence of S9 mix (see Table 7 and Table 8 in attached document)

Cultures were exposed to Gamma-Nonalactone at concentrations from 100 to 800 mg/mL. No precipitate was observed by eye at the end of treatment. Cultures exposed to Gamma-Nonalactone at concentrations from 100 to 550 mg/mL were assessed for determination of mutation frequency. RTG values from 131 to 17% were obtained relative to the vehicle control. There were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum (214 x 10-6) of the mean concurrent vehicle control mutant frequency (88 x 10-6) and the GEF (126 x 10-6), within acceptable levels of toxicity.

The positive control, methyl methanesulphonate, induced an acceptable increase in mutation frequency and an acceptable increase in the number of small colony mutants.

Applicant's summary and conclusion

Conclusions:
Under the test conditions, Gamma-Nonalactone did not demonstrate mutagenic potential in this in vitro cell mutation assay in the absence and presence of metabolic activation.
Executive summary:

Gamma-Nonalactone was tested for mutagenic potential in an in vitro mammalian cell mutation assay according to OECD 476 guideline and in compliance with GLP. This test system is based on detection and quantitation of forward mutation in the subline 3.7.2c of mouse lymphoma L5178Y cells, from the heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient genotype (TK-/-).

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

Gamma-Nonalactone was found to be soluble at 156.2 mg/mL in acetone. A final concentration of 1562 mg/mL (10 mM), dosed at 1% v/v, was used as the maximum concentration in the preliminary toxicity test, in order to test up to the standard limit concentration within this test system as recommended in the regulatory guidelines. 

Toxicity was observed in the preliminary toxicity test. Following a 3 hour exposure to Gamma-Nonalactone at concentrations from 3.1 to 1562 mg/mL, relative suspension growth (RSG) values were between 132 and 0% and between 109 and 2% in the absence and presence of S9 mix respectively. Following a 24 hour exposure in the absence of S9 mix RSG vales were between 104 and 0%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG).

Following 3 hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency (93 x 10-6and 88 x 10-6, respectively) and the Global Evaluation Factor (GEF) of 126 x 10-6, within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 hour treatment in the absence and in the presence of S9 mix were 550 and 1050 mg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 23 and 11% respectively.

In the 24 hour treatment, the maximum concentration assessed for mutant frequency was 550 mg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control (88 x 10-6) mutant frequency and the GEF of 126 x 10-6, within acceptable levels of toxicity. The RTG was reduced to 17%.

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

It was concluded that Gamma-Nonalactone did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.