2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity in bacteria: negative (OECD 471) Gene mutation in mammalian cells: negative (OECD 476) Chromosome aberration: negative (OECD 473)

Link to relevant study records

Reference

Reference 1

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:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP- and guideline compliant

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

hprt locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: selected in HAT medium to reduce the number of mutants before the test started

Metabolic activation:

with and without

Metabolic activation system:

S9 supernatant and S9 cofactor solution (below) to result in a final protein concentration of 0.75 mg/mL in the cultures 8 mM MgCl2 33 mM KCl 5 mM glucose-6-phosphate 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

Test concentrations with justification for top dose:

The test concentrations below are in µg/mL
Experiment I
without S9 mix* 6.3 12.5 25.0 50.0 600.0 800.0 1200.0
with S9 mix* 6.3 12.5 25.0 50.0 600.0 800.0 1200.0
Experiment II
without S9 mix** 10.0 20.0 40.0 80.0 320.0 480.0 640.0
With S9 mix* 10.0 20.0 40.0 80.0 640.0 960.0 1280.0
Experiment III
without S9 mix** 100.0 200.0 400.0 600.0 800.0 1000.0
* 4 hours treatment
** 24 hours treatment

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells.

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The study was performed to investigate the potential of ASA to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.
The assay was performed in three independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. Since the recommended cytotoxic range of a relative cloning efficiency of approximately 10 - 20% was not covered in the second experiment without metabolic activation a repeat experiment was performed at higher concentrations (experiment III). The treatment time of experiment III was 24 hours without metabolic activation.
The highest concentration of the test item in the pre-experiment was 5 µL/mL. The concentration range of the main experiments was limited by cytotoxic effects and the solubility of the test item in aqueous medium.
Appropriate reference mutagens, used as positive controls, were used to demonstrate the sensitivity of the test item and the activity of the metabolic activation system.

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours (+/- S9) 1st experiment / 4 hours (+S9) and 24 hours (-S9) 2nd experiment / 24 hours (-S9) 3rd experiment
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): about 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): about 15 days

SELECTION AGENT (mutation assays): 6-Thioguanine
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 3 - 5 x10^5 cells per flask (concentration, +/-S9, experiment, replicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: determined with a separate assay

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: no
- Other:

OTHER:

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-muta¬genic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation fre¬quency at least at one of the concen¬trations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
experimental group p-value
experiment I, culture I without S9 mix 0.254
experiment I, culture II without S9 mix 0.079
experiment I, culture I with S9 mix 0.670
experiment I, culture II with S9 mix 0.955
experiment II, culture I without S9 mix 0.308
experiment II, culture II without S9 mix 0.094
experiment II, culture I with S9 mix 0.853
experiment II, culture II with S9 mix 0.582

Species / strain:

Chinese hamster lung fibroblasts (V79)

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. The pH was checked in the solvent control (pH 7.45) and in the highest concentration (pH 7.04)
- Effects of osmolality: none. Osmolarity was checked in the solvent control (391) and in the highest concentration (322)
- Evaporation from medium: none: Vapor pressure is very low
- Water solubility: ASA is a uvcb substance. There are at least some water-insoluble fractions at the higher test concentrations.
- Precipitation: The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) before the test item was removed. Precipitation was observed by the unaided eye down to the lowest concentration of 0.039 µL/mL with and without metabolic activation following 4 hours treatment. After 24 hours treatment (without metabolic activation) precipitation occurred at 0.078 µL/mL and above.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency

COMPARISON WITH HISTORICAL CONTROL DATA: The parameters of this study are all in the range of the historical controls

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first and second experiment with and without metabolic activation the cultures at the lowest concentration with metabolic activation and at the two lowest concentrations without metabolic activation were not continued since a minimum of only four analysable concentrations is required by the guidelines. The maximum concentration with metabolic activation was not continued in both experiments due to exceedingly severe cytotoxic effects. In experiment III the three highest concentrations were not continued for the same reason.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results: negative

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, ASA is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The test item ASA was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The study was performed in three independent experiments, using identical general experimental procedures. In the main experiments of this study (with and without S9 mix) the range of the solvent controls was from 2.6 up to 30.1 mutants per 10⁶cells; the range of the groups treated with the test item was from 0.0 up to 50.8 mutant colonies per 10⁶cells.

EMS(0.15 mg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, ASA is considered to be non-mutagenic in this HPRT assay.

Reference 2

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:

2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study in compliance with GLP and guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

primary culture, other: lymphocytes

Details on mammalian cell type (if applicable):

Blood samples were obtained from healthy donors not receiving medication. For this study, blood was collected from a female donor (35 years old) for the first and second experiment and from a 46 year-old male donor for Experiment II. Blood samples were drawn by venous puncture and collected in heparinized tubes. Short-term cultures of human lymphocytes were stimulated to divide by the addition of a phytohaemagglutinin, PHA) to the culture medium. Mitotic activity began at about 40 hours after PHA stimulation and reached a maximum at around 3 days. The
chromosome constitution remained diploid during short-term culture.
Treatments commenced 50 - 80 hours after culture initiation when the cells were actively proliferating At preparation time 22 hours, a minimum of three cultures treated with separated concentrations of the test item were evaluated for the potential to induce structural chromosomal aberrations.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Experiment 1
DMSO 0.5 % without S9 4 / 18 hrs
Pos. control 825.0 µg without S9 4 / 18 hrs
ASA 22.7 µg without S9 4 / 18 hrs
ASA 39.8 µg without S9 4 / 18 hrs
ASA 213.2 µg without S9 4 / 18 hrs
ASA 373.2 µg without S9 4 / 18 hrs
DMSO 0.5 % with S9 4 / 18 hrs
Pos. control 22.5 µg with S9 4 / 18 hrs
ASA 22.7 µg with S9 4 / 18 hrs
ASA 39.8 µg with S9 4 / 18 hrs
ASA 121.9 µg with S9 4 / 18 hrs
ASA 213.2 µg with S9 4 / 18 hrs

Experiment 2
DMSO 0.5 % without S9 22 / 0 hrs
Pos. control 770.0 µg without S9 22 / 0 hrs
ASA 13.0 µg without S9 22 / 0 hrs
ASA 22.7 µg without S9 22 / 0 hrs
ASA 69.6 µg without S9 22 / 0 hrs
ASA 121.9 µg without S9 22 / 0 hrs
DMSO 0.5 % with S9 4 / 18 hrs
Pos. control 30.0 µg with S9 4 / 18 hrs
ASA 29.8 µg with S9 4 / 18 hrs
ASA 52.2 µg with S9 4 / 18 hrs
ASA 279.9 µg with S9 4 / 18 hrs
ASA 489.8 µg with S9 4 / 18 hrs

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; ASA was added to the test medium dissolved in DMSO. Cultures were treated with test medium.

DURATION
- Preincubation period: 72 hours
- Exposure duration: 4 hours or 18 hours or 22 hours, respectively
- Expression time (cells in growth medium): 18 hours, 4 hours or 0 hours, respectively
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, for 3 hours prior fixation
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 per concentration, each with and without S9 and experiment I and II

NUMBER OF CELLS EVALUATED: 2x100 metaphases per concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (main test) and cloning efficiency (range finder)

OTHER EXAMINATIONS:
- Determination of polyploidy: visual (100x microscope) counting of centromers. If multiple copies of the haploid chromosome number (other than diploid) are scored then the count is recorded and the cell classified as polyploid.
- Determination of endoreplication: visual (100x microscope). If the chromosomes are arranged in closely apposed pairs, i.e. 4
chromatids instead of 2, the cell is scored as endoreduplicated.

Evaluation criteria:

Only metaphases with 46 ± 1 centromer regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in
mitosis) was determined

A test item is classified as non-mutagenic if the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, excluding gaps). and no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as mutagenic if the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, excluding gaps) and either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test item can be classified as aneugenic if the number of induced numerical aberrations is not in the range of our historical control data
(0.0 – 0.8 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant increase in the pH value was observed (e.g. Exp. I: solvent control: pH 7.4 versus pH 7.5 at 3500 µg/mL).
- Effects of osmolality: No relevant increase in the osmolarity was observed (e.g. Exp. I: solvent control: 378 mOsm, 330 mOsm at 3500 µg/mL).
- Evaporation from medium: not expected due to very low vapor pressure
- Water solubility: The test material is uvcb. Water solubility is not defined.
- Precipitation: In Experiment I, visible precipitation of the test item in the culture medium was observed at 39.8 µg/mL and above in the absence of S9 mix and at 69.6 µg/mL and above in the presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence of S9 mix at 22.7 µg/mL and above and at 52.2 µg/mL and above in the presence of S9 mix.

- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the mutagenicity assay. The pre-test phase was performed with 10 concentrations of the test item and a solvent and positive control. All cell cultures were set up in duplicate. Exposure times was 4 hrs (with and without S9 mix). The preparation interval was 22 hrs after start of the exposure.

COMPARISON WITH HISTORICAL CONTROL DATA: Comparison with historical control is a prerequisite for acceptance of the study. To be valid, the study has to meet the following criteria:
a) The number of aberrations found in the solvent controls falls within the range of historical laboratory control data range: 0.0 - 4.0 % aberrant cells, excluding gaps.
b) The positive control substances should produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory’s historical control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

In Experiment I, visible precipitation of the test item in the culture medium was observed at 39.8µg/mL and above in the absence of S9 mix and at 69.6µg/mL and above in the presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence of S9 mix at 22.7µg/mL and above and at 52.2µg/mL and above in the presence of S9 mix. Cytotoxic effects were observed in all experimental parts of this study. In detail, clearly reduced mitotic indices occurred at the highest evaluated concentrations in Experiment I (54.3 % of control in the absence of S9 mix, 50.4 % of control in the presence of S9 mix) and in Experiment II (21.1 % of control in the absence of S9 mix, 45.9 % of control in the presence of S9 mix). In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, excluding gaps) were close to the range of the solvent control values (1.5 – 3.0 % aberrant cells, excluding gaps) and within the range of the laboratory´s historical solvent control data: 0.0 - 4.0 % aberrant cells, excluding gaps. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. In both experiments, either EMS (770 or 825µg/mL) or CPA (22.5 or 30.0µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:

Interpretation of results: negative

Under the experimental conditions reported, the test item did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, C16/18 Alkenyl succinic anhydride is considered to be non-clastogenic in this chromosome aberration test when tested up to cytotoxic and precipitating concentrations.

Executive summary:

C16/18 Alkenyl succinic anhydride, dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytes in vitro in two independent experiments in vitro, with and without S9 mix. In each experimental group two parallel cultures were analysed. Per culture 100 metaphase plates were scored for structural chromosomal aberrations. The highest applied concentration in the pre-test on toxicity (3500 µg/mL of the test item) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation.

In the absence and presence of S9 mix, cytotoxicity was observed at the highest evaluated concentrations being far in the range of test item precipitation. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Reference 3

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:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP- and guideline compliant

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his; trp

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from liver of phenobarbitaland β-naphthoflavone rats

Test concentrations with justification for top dose:

1st Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 0.2; 1; 5; 25 and 50 μg/plate

2nd Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 20.8; 104; 520; 2 600 and 5 200 μg/plate

3rd Experiment
Strain: TA 100
Doses: 0; 31.3; 62.5; 125; 250 and 500 μg/plate

4th Experiment
Strains: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA
Doses: 0; 20.8; 104; 520; 2 600 and 5 200 μg/plate (TA 1535, TA 1537, TA 98 and E. coli)
0; 2; 10; 50; 250 and 500 μg/plate (TA 100)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: limited solubility of the test item in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD),9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation;

1. Standard plate test
The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et al.
• Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar:
980 mL purified water
20 mL Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

• Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds. The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M.H.L. and Muriel, W.J. ( 5), with the exception of the amino acid solution, which has previously been added to the soft agar:
300 mL solution B (agar)
100 mL solution A (saline solution)
8 mL solution C (glucose solution)
10 mL solution D (casein solution)
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

2. Preincubation period: 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.

Evaluation criteria:

1. Mutagenicity
Individual plate counts, the mean number of revertant colonies per plate and the standard deviations were given for all dose groups as well as for the positive and negative (vehicle) controls in all experiments. In general, five doses of the test item are tested with a maximum of 5 mg/plate, and triplicate plating is used for all test groups at least in the 1st Experiment. Dose selection and evaluation as well as the number of plates used in repeat studies or further experiments are based on the findings of the 1st Experiment.
2. Toxicity
Toxicity detected by a
• decrease in the number of revertants
• clearing or diminution of the background lawn (= reduced his- or trp- background growth)
• reduction in the titer is recorded for all test groups both with and without S9 mix in all experiments and indicated in the tables.
3. Acceptance criteria
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain
• The sterility controls revealed no indication of bacterial contamination.
• The positive control items both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above
• The titer of viable bacteria was ≥ 108/mL.

Statistics:

none

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from about 25 μg/plate onward in the standard plate tests; from about 250 μg/plate onward in the preincubation assay

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ or trp+
revertants, reduction in the titer) was observed in the standard plate tests depending on the
strain and test conditions from about 25 μg/plate onward.
In the preincubation assay bacteriotoxicity (reduced his- background growth, decrease in the
number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain
and test conditions from about 250 μg/plate onward.

SOLUBILITY
Test item precipitation was found at 5 200 μg/plate onward with and without S9 mix.

MUTAGENICITY
T
he test item did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in several experiments carried out independently of each other (standard plate test and preincubation assay).
Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

Conclusions:

Interpretation of results: negative

It is concluded that ASA is not a mutagenic test item in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The test item was tested for mutagenicity in the Salmonella typhimurium / Escherichia coli reverse mutation assay according to OECD 471 and GLP. The substance was tested in the standard plate test and in the preincubation test both with and without the addition of a metabolizing system (S9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. As a result, no increase in the number of revertants was found, so that the substance was not found to be mutagenic under the condition of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The test item was tested for mutagenicity in the Salmonella typhimurium / Escherichia coli reverse mutation assay according to OECD 471 and GLP. The substance was tested in the standard plate test and in the preincubation test both with and without the addition of a metabolizing system (S9 mix) obtained from rat liver using the Salmonella strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA. As a result, no increase in the number of revertants was found, so that the substance was not found to be mutagenic under the condition of the test.

The substance was assessed for its potential to induce gene mutations at the HPRT locus in a study performed according to OECD 476 and GLP using V79 cells of the Chinese hamster. In the main experiments of this study (with and without S9 mix) the range of the groups treated with the test item was from 0.0 up to 50.8 mutant colonies per 10E6cells. In conclusion, the test item did not induce gene mutations at the HPRT locus in V79 cells under the experimental conditions reported. Therefore, the test substance is considered to be non-mutagenic in this HPRT assay.

The potential to induce structural chromosomal aberrations in human lymphocytes was assessed in a study in accordance with OECD 473 and GLP in two independent experiments, each with and without S9 mix. The highest applied concentration in the pre-test on toxicity (3500 µg/mL of the test item) was chosen with regard to the solubility properties of the test item. Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation. In the absence and presence of S9 mix, cytotoxicity was observed at the highest evaluated concentrations being far in the range of test item precipitation. In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures. Under the experimental conditions reported, the test item did not induce structural chromosomal aberrations as determined by the chromosome aberration test in human lymphocytes in vitro. Therefore, the test substance is considered to be non-clastogenic in this chromosome aberration test when tested up to cytotoxic and precipitating concentrations.

Justification for selection of genetic toxicity endpoint
Guideline study according to GLP

Justification for classification or non-classification

The available data are conclusive but not sufficient data for classification with regard to genetic toxicity in accordance with the CLP Regulation (EC) No 1272/2008.