Pyrrolidinium, 1-[[(6R,7R)-7-amino-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl]-1-methyl-, chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test-item was not mutagenic in the Ames test and in the in vitro chromosome aberration assay.

The test item was not mutagenic in the in vitro mammalian cell gene mutation test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline Study with GLP

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his- (S. typhimurium)

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

other: S. typhimurium TA97a

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from Aroclor 1254 induced microsomes of rat liver

Test concentrations with justification for top dose:

The test substance was dissolved in water. The following concentrations were tested:
7, 21, 62, 185, 556 and 1667 µg per plate in the first experiment.
2.3, 7, 21, 62, 185 and 556 µg per plate in the second experiment.

Vehicle / solvent:

Deion. water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene; 7,12-Dimethylbenz[a]anthracene ; 1,8-Dihydroxy-anthraquinone; 2-Nitrofluorene; Sodium azide; 4-Nitro-o-phenylenediamine; t-Butyl-hydroperoxide

Details on test system and experimental conditions:

A preliminary toxicity test was performed with strain TA100. The test substance was toxic to the bacteria at 5000 and 1667 µg/plate, resulting in a missing bacterial background lawn. Therefore 1667 µg/plate was chosen as highest concentration which could be in the toxic range and 6 concentrations were tested. The concentrations for the second experiment were changed due to the results of the first experiment: The test substance was toxic to the bacteria at 1667 µg/plate, resulting in a missing bacterial background lawn or a reduced number of colonies, and at 556 µg/plate resulting in a reduced number of colonies. Therefore the concentrations were decreased one step.

The exposure for the first experiment was performed according to the 'Plate Incorporation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state.
For each sample the following solutions were combined:
• 0.1 mL of the overnight culture of the bacteria,
• 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 mL of the appropriate test- or reference substance solution and
• 2 mL of top agar.
The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).


The exposure for the second experiment was performed according to the 'Preincubation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate with preceding incubation in the liquid state.
For each sample the following solutions were combined:
• 0.1 mL of the overnight culture of the bacteria,
• 0.5 mL of S9-mix (or phosphate buffered saline for samples without metabolic activation),
• 0.1 mL of the appropriate test- or reference substance solution.
The solutions were preincubated for 20 minutes at 37 °C using a shaker, afterwards combined with 2 mL of top agar and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days).

Evaluation criteria:

The criteria for a positive result are:
A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations:
• For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 2½ fold of the amount of the spontaneous revertants.
• For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 1.67 fold of the amount of the spontaneous revertants.

Statistics:

Means and standard deviations were calculated for the number of mutants in every concentration group.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

other: S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle 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

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle 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

Positive controls validity:

valid

Additional information on results:

Solubility:
No precipitation of the test substance was seen in any of the concentration groups.

Toxicity
In the preliminary test the test substance was toxic to the bacteria at 5000 and 1667 µ/plate, resulting in a missing bacterial background lawn.
In the main test the test substance was again toxic to the bacteria at 1667 µ/plate, resulting in a missing bacterial background lawn or a reduced number of colonies, and at 556 µg/plate resulting in a reduced number of colonies.

Mutagenicity
There was no increase in the number of mutants in any of the tested bacterial strains at any of the tested concentrations. The addition of an external metabolising system did not change these results.

Properties of the bacteria:
The used strains of Salmonella typhimurium showed the expected genetic properties and were sensitive against several mutagenic chemicals. The numbers of spontaneous revertants were comparable with the historic control data for the negative controls.

Positive control substances:
All positive control substances increased the mutation frequency to more than the threshold values stated above. As 2-aminoanthracene, 1 ,8-dihydroxy-anthraquinone and 7, 12-dimethyl-benz[a]anthracene require metabolic activation for mutagenicity, the results of these substances demonstrate also the efficiency of the metabolising system.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

According to these results, MPI-ACA is not mutagenic in the Ames test with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 with and without an external metabolising system up to 1667 µ/plate, which is the limit of toxicity.

Executive summary:

MPI-ACA was tested for mutagenic activity with the "Salmonella typhimurium Reverse Mutation Test" (Ames Test). The study was conducted in accordance with the OECD-guideline 471 and directive 2000/32/EC, part 8.13/14.

Two independent experiments were performed with and without an external metabolising system. The first experiment was performed according to the "direct plate incorporation method", the second experiment according to the ''preincubation method". As test system the bacterial strains Salmonella typhimurium TA97a, TA98, TA100, TA1 02 and TA1535 were used. Negative and positive controls were included.

Results:

Positive controls: All positive control groups showed significantly increased mutation frequencies which demonstrate the sensitivity of the test system.

Toxicity: The test substance was toxic to the bacteria at 1667 µg/plate, resulting in a missing bacterial background lawn or a reduced number of colonies, and at 556 µg/plate resulting in a reduced number of colonies.

Solubility: No precipitation of the test substance was seen in any of the concentration groups.

Mutagenicity: In none of the concentrations tested and with none of the strains used an increase of the mutation frequency to more than the threshold values (250 % of the controls for strains TA98 and TA 1535, 167 % of the controls for strains TA97a, TA100 and TA102) was obtained. Metabolic activation did not change these results.

According to these results, MPI-ACA is not mutagenic in the Ames test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2014

Deviations:

yes

Remarks:

Ethyl methanesulfonate was used as the positive control.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammalian chromosome aberration test.

Target gene:

Not applicable.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Lot. No.: 10H016
Supplier: ECACC (European Collection of Cells Cultures)
The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations. These cells were chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14 h).
The cell stocks were kept in a freezer at -80 +/- 10 °C. Checking for mycoplasma infections was carried out. Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA x 4 Na) solution was used for cell detachment to subculture. The laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 °C in an incubator with a humidified atmosphere, set at 5 % CO2. The V79 cells for this study was grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphoptericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 3 and 20 hours treatments with test item, negative and positive controls, the serum content was reduced to 5%.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rodent S9 mix

Test concentrations with justification for top dose:

Experiment A:
Without S9 mix: 0 - 500 - 1000 - 2000 µg/mL (3 h exposure; sampling 20 h after start of exposure).
With S9 mix: 0 - 500 - 1000 - 2000 µg/mL (3 h exposure; sampling 20 h after start of exposure).

Experiment B:
Without S9 mix: 0 - 125 - 250- 500 µg/mL (20 h exposure; sampling 20 h after start of exposure)
With S9 mix: 0 - 125 - 250- 500 µg/mL (20 h exposure; sampling 28 h after start of exposure)
With S9 mix: 0 - 500 - 1000 - 2000 µg/mL (3 h exposure; sampling 28 h after start of exposure).

Selection of doses according to the results of the preliminary test.

Vehicle / solvent:

The test item solutions were prepared in the testing laboratory using DME (Dulbecco’s Modified Eagle’s) medium as solvent.
This vehicle is compatible with the survival of the V79cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DME

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Components of Media:
Name: DME (Dulbecco’s Modified Eagle’s) medium
Supplier: Sigma-Aldrich, Germany
Name: Fetal Bovine Serum
Supplier: Sigma-Aldrich, Germany
Name: Antibiotic-antimycotic
Supplier: Sigma-Aldrich, Germany

Rat Liver S9 Fraction
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH, Germany; manufacturer: MOLTOX INC., USA. Certificate of Analysis was obtained from the supplier.

Pre-test for Cytotoxicity (Concentration selection)
The pre-experiments on solubility of the test item (non GLP) and GLP Pre-test for Cytotoxicity (Concentration selection) were performed. In order to determine the treatment concentrations of test item in the cytogenetic study a dose selection (cytotoxicity assay) was performed. During the cytotoxicity assay the cells were seeded into 92 x 17 mm dishes (for tissue cultures in TC sterile quality) at 5 x 105 cells each and were incubated for 24 hours in 10 mL of DME (Dulbecco’s Modified Eagle’s) medium containing 10 % foetal bovine serum. After 24 hours the cells were treated using increasing concentrations of test item in the absence or presence of S9 mix (50 mg/mL) and were incubated at 37 °C for 3 hours. After treatment the cultures were washed with DME medium and covered with DME (Dulbecco’s Modified Eagle’s) medium containing 10 % foetal bovine serum. Cell counts were performed after 20 hours (approximately 1.5 normal cell cycles from the beginning of treatment). The cells were counted using a Bürker chamber. Additional groups of cells were treated for 20 hours without metabolic and for 3 hours with metabolic activation, with cell counts conducted after 20 hours (without S9 mix only) and 28 hours (without and with S9 mix). Additionally, 4 cultures were set up for determining the initial cell count. At harvest the cells were trypsinised, collected and cell counts were determined. Based on the cell counts Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. The volume of culture medium was 5 mL/dish for each per group. The results obtained were used for dose selection of the test item used in the Chromosome Aberration Assays (Experiments A and B). In addition, the pH and osmolality was measured in this pre-test.

Chromosome Aberration Assays:
Experiment A:The test item was dissolved in DME (Dulbecco’s Modified Eagle’s) medium for the treatment (stock solution: 100 mg/mL). The appropriate amount of this stock solution was completed with DME (Dulbecco’s Modified Eagle’s) medium to reach the constant volume. The constant volume was diluted with medium to obtain the examination concentrations. Duplicate cultures were used at each concentration and the negative control cultures as well as the positive controls for treatment without and withS9 mix. 5 x 105 cells were set up at each group. The culture medium of exponentially growing cell cultures was replaced with medium containing the test item. The exposure period was 3 hours. The exposure period was followed by washing the cells with DME (Dulbecco’s Modified Eagle’s) medium and then growth medium was added. Sampling was made at 20 hours after treatment start (approximately 1.5 normal cell cycles from the beginning of treatment). For concurrent measures of cytotoxicity for all treated and negative control cultures, 5 x 105 cells were set up.
Experimnet B: In the cytogenetic Experiment B the exposure period without metabolic activation was 20 hours. The exposure period with metabolic activation was 3 hours.
Experiment B, as Experiment A, included a concurrent S9 non-activated and S9 activated positive and negative control. For each group 5 x 105 cells/dish cells were seeded. Sampling was made at 1.5 cell cycles (20 hours, without S9 mix only) and at approximately 2 normal cell cycles (28 hours, without and with S9 mix) from the beginning of treatment to cover a potential mitotic delay.

Measurement of pH and Osmolality
The pH value and osmolality of negative (solvent) control and test item treatment solutions (for every treatment concentrations) were measured in the Pre-test for Cytotoxicity (Concentration selection) and in the Chromosome Aberration Assay.

Preparation of Chromosomes
Cell cultures were treated with Colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.

Analysis of Methaphase Cells
All slides were independently coded before microscopic analysis and scored blind. 300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration as well as the negative and positive controls and were equally divided among the duplicates (150 metaphases/slide). Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally, the number of polyploid and endoreduplicated cells were scored. The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983.

Rationale for test conditions:

See above.

Evaluation criteria:

– Different types of structural chromosome aberrations are listed, with their numbers and frequencies for experimental and control cultures.
– Gaps were recorded separately and reported, but generally not included in the total aberration frequency.
– Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment (s) were recorded.
– Individual culture data were summarised in tabular form.
– There were no equivocal results in this study.
– pH and Osmolality data were summarised in tabular form.

Interpretation of Results
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if:
– at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– the increase is dose-related when evaluated with an appropriate trend test,
– any of the results are outside the distribution of the laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative because:
– none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
– there is no concentration-related increase when evaluated with an appropriate trend test

Statistics:

For statistical analysis CHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

In Experiment A, MPI-ACA did not induce an increase in the number of cells with aberrations without gaps at any examined concentration, neither in the absence nor in the presence of metabolic activation. (Tables 3, 4 and 8). There were no statistically significant differences between test item treatment and control groups and no dose-response relationship was noted.
In Experiment B, MPI-ACA was examined without S9 mix, over a long treatment period and the sampling was made at approximately 1.5 cell cycles (20 hours after treatment start). The cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent solvent and historical controls.
There was no increase in the number of cells with aberrations without S9 mix following exposure over a long treatment period of 20 hours and sampling at approximately 2 cell cycles (28 hours after treatment start).
A 3-hour treatment in the presence of S9 mix with 28-hour harvest from the beginning of treatment did not cause an increase in the number of cells with structural chromosome aberrations without gaps compared to the concurrent solvent and historical controls (Tables 5, 6, 7 and 9).
In Experiment A and in Experiment B no statistically significant differences between test item treatment and controls (solvent and historical) groups and no dose-response relationships were noted.
No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of the test item.

There was no precipitation of the test item at any dose level tested.
pH and osmolality values of control and test item treatment solutions were measured. In Experiments A and B no significant differences between test item treatment and control groups were observed.
In the concurrent negative control group the percentage of cells with structural aberration(s) without gap was less than 5 %, confirming the suitability of the cell line used.
The number of aberrations found in the solvent controls was compatible with the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and Cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data (Tables 3, 4, 5, 6 and 7). Thus, the study is considered valid.

Conclusions:

The test item, with and without mammalian metabolic activation system, did not induce structural chromosome aberrations in Chinese Hamster lung cells. The test item is not clastogenic in this system.

Executive summary:

A Chromosome Aberration Assay in V79 cells was performed. The test item was dissolved in DMSO and then in Dulbecco’s Modified Eagle’s medium and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study. In two independent experiments (both run in duplicate with concurrent negative and positive controls) at least 300 (150-150) well-spread metaphase cells were analysed at concentrations and treatment (exposure)/sampling (expression) intervals given below:

Experiment A:

Without S9 mix: 0 - 500 - 1000 - 2000 µg/mL  (3 h exposure; sampling 20 h after start of exposure).

With S9 mix:  0 - 500 - 1000 - 2000 µg/mL (3 h exposure; sampling 20 h after start of exposure).

Experiment B:

Without S9 mix: 0 - 125 - 250- 500 µg/mL (20 h exposure; sampling 20 h after start of exposure)

With S9 mix: 0 - 125 - 250- 500 µg/mL (20 h exposure; sampling 28 h after start of exposure)

With S9 mix: 0 - 500 - 1000 - 2000 µg/mL (3 h exposure; sampling 28 h after start of exposure).

Results:

In both experiments, no statistically significant differences between treatment and concurrent solvent control groups and no dose-response relationships were noted. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

In the concurrent negative control group the percentage of cells with structural aberration(s) without gap was less than 5 %, confirming the suitability of the cell line used. The number of aberrations found in the solvent controls was compatible with the historical laboratory control data.

The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and Cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

Principles of method if other than guideline:

Mutant cells deficient in Hprt enzyme activity in the HPRT test are resistant to the cytostatic effects of the purine analogue 6-thioguanine (TG). The Hprt proficient cells are sensitive to TG, which causes the inhibition of cellular metabolism and halts further cell division. Thus, mutant cells are able to proliferate in the presence of TG, whereas normal cells, which contain the Hprt enzyme, are not.

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: HPRT test

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase enzyme locus.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO K1: Sub-line (K1) of Chinese hamster ovary cell line CHO
Lot. No.: 12G006
Supplier: ECACC (European Collection of Cell Cultures)

Each batch of frozen cells was purged of HPRT mutants and was free for mycoplasma infections. For each experiment the cells were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air. Growing cells were subcultured in an appropriate number of flasks. The CHO K1 cells for this study were grown in Ham's F12 medium (F12-10) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 5 treatments with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 % (F12-5). The selection medium for TG resistant mutants contained 3.4 μg/mL 6-thioguanine (6-TG) (EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of phenobarbital and β-naphthoflavone induced rat liver.

Test concentrations with justification for top dose:

5-hour treatment period without S9-mix: 250, 500, 1000, 1500 and 2000 μg/mL
5-hour treatment period with S9-mix: 250, 500, 1000, 1500 and 2000 μg/mL
The concentrations were chosen based on the cytotoxicity and the maximum recommended concentration.

Vehicle / solvent:

The test item was prepared in Ham's F12 medium and diluted prior to treatment.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Ham's F12 medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

A pre-test on toxicity was performed to select treatment concentrations for the mutation assay.

A 5-hour treatment in the presence and absence of S9-mix was performed. For the 5-hour treatment, 5 x10E6 cells were placed in sterile dishes and incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2. Duplicate cultures were used at each test item concentration, for negative (solvent) controls and the positive controls for treatment without and with S9-mix. On the day of treatment the culture medium of exponentially growing cell cultures were replaced with medium (F12-5) containing the test item. The exposure period was 5 hours. Following the exposure period the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours. After the 19-hour incubation period, cells were washed twice with F12-10 medium and suspended by treatment with trypsin-EDTA solution and counted using a Bürker chamber. Solubility of the test item in the cultures was assessed by the naked eye, at the beginning and end of treatment. In samples where sufficient cells survived, cell number was adjusted to 10E5 cells/mL. Throughout the expression period, cells were transferred to dishes for growth or diluted to be plated for survival.

The pH and osmolality of the negative (solvent) control and test item solutions were determined in Experiment 1 and Experiment 2.
Plating for survival: Following adjustment of the cultures to 10E5 cells/mL, samples from these cultures were diluted to 40 cells/mL. A total of 5 mL (200 cells/dish) of the final concentration of each culture was plated into 3 parallel dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days for growing colonies. Then, colonies were fixed with methanol, stained with Giemsa and counted. Survivals were assessed by comparing the cloning efficiency of the test item treated groups to the negative (solvent) control.
Expression of the mutant phenotype: During the phenotypic expression period the cultures were subcultured. Aliquots of approximately 2x10E6 cells were taken on days 1, 3, 6, and evaluated on day 8.
Selection of the mutant phenotype: At the end of the expression period, cultures from each dose level were adjusted to 2 x 10E5 cells / dish ( 4 x five dishes) in selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 μg/mL of thioguanine (6-TG).
Plating for viability: At the end of the expression period cell number in the samples were adjusted to 2 × 10E5 cells/mL. Cells were plated in 3 parallel dishes (diameter is approx. 60 mm) for a viability test as described in “Plating for Survival“ section for the survival test.
Fixation and staining of colonies: After the selection period, the colonies were fixed with methanol for five minutes, stained with Giemsa and counted for either mutant selection or cloning efficiency determination.

Rationale for test conditions:

See above.

Evaluation criteria:

Calculation of mutation frequency: The mutation frequency was calculated by dividing the total number of mutant colonies by the number of cells selected (10E6 cells: 5 plates at 2 x 10E5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection (viability), and was expressed as 6-TG resistant mutants per 10E6 clonable cells.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

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:

The test item, was dissolved in Ham's F12 medium. A clear solution was obtained up to a concentration of 100 mg/mL. For examined test item concentrations no precipitation in the medium was noted. No relevant changes in pH or osmolality were found after treatment with the test item.

On Day 1, there was very clear evidence of toxicity with the test item in presence of metabolic activation (S9 mix) when compared to the negative (solvent) controls, confirming the response seen in the dose selection cytotoxicity assays. The Day 8 cloning efficiency data indicate that in general the cells had recovered during the expression period.
Low toxicity was observed in treatment groups when compared to the negative (solvent) controls, in the absence of the metabolic activation, confirming the response seen in the dose selection cytotoxicity assays. The test item MPI-ACA was low cytotoxic in the examined concentrations without metabolic activation.
The mutation data showed no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no significant differences between treatment and control groups and no dose-response relationships were noted. All values were within the range of the laboratory historical solvent control data and no dose-related increase was observed in any of the cultures.

The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with Ethyl methanesulfonate (1.0 μL/mL) and 7,12-Dimethyl benz[a]anthracene (20 μg/mL). The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory. Thus, the study is considered valid.

Conclusions:

The test item was not mutagenic in this in vitro mammalian cell gene mutation test.

Executive summary:

An in vitro genotoxicity test, the HPRT-test, was performed according to the OECD guuideline 476. The test item, applied up to the maximum recommended concentration (2000 μg/mL) concentration with and without metabolic activation system over a 5 hour treatment period did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control).

It is concluded that the test item was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Conclusive but not sufficient for classification.