17-acetoxy-1β,2β-methanopegna-4,6-diene-3,20-dione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation (bacterial reverse mutation assay / plate incorporation, GLP, OECD TG 471): negative with and without metabolic activation [Report AN51]

Gene mutation (bacterial reverse mutation assay / pre-incubation, GLP, OECD TG 471): negative with and without metabolic activation (Report AY91]

Chromosomal abberation (GLP, OECD TG 473): negative in first experiment [Report AZ10]

Chromosomal abberation (GLP, OECD TG 473): negative in second experiment [Report AZ08]

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

June 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Justification for type of information:

Second independent experiment (in addition to study TXST19980072) with different harvesting times

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 July 1997

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not specified

Species / strain / cell type:

lymphocytes: human primary cells

Details on mammalian cell type (if applicable):

- Type and identity of media: MCCoy's 5a medium
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver homogenate
Type and composition of metabolic activation system:
S9 (batch no. 110997), derived from male Wistar rats pretreated with phenobarbital and ß-naphthoflavone, protein content 26.8 mg/mi, was prepared by RCC-CCR (Cytotest Cell Research, Roßdorf, Germany). The components of the standard 89 mix were 12.2% (viv) 89, 4 mmol/l NADP, 5 mmol/l glucose-6-phosphate, 8 mmol/I MgCI2, 34.3 mmol/l KCI and 50 mmol/l sodium phosphate buffer, pH 7.4.

Test concentrations with justification for top dose:

10, 100 and 125 µg/ml in the assay without S9 mix (first harvesting time, 19 hours after a
4-hour treatment), whereby the mitotic index was reduced by 33% at the highest
concentration.

125 µg/ml in the assay without S9 mix (second harvesting time, 40 hours after a 4-hour
treatment).

10, 75 and 125 µg/ml in the assay with S9 mix (harvesting time, 19 hours after a 4-hour
treatment)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of ZK 5690 was tested in DMSO, yielding a solubility of > 12.5 mg/ml. But when this stock solution was added at 1% to tissue culture medium containing 15% (vIv) FCS precipitates of the test compound were visible starting at ca. 50 µg/ml.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: -S9 mix: triaziquone

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: One

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 h
- Exposure duration/duration of treatment: without S9 mix: 19h first harvesting, 40 h second harvesting; with S9 mix: 19 h harvesting


FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure. To arrest the cells in the metaphase, colcemide (final concentration 0.08 µg/mL) was added ca. 3.5 h before the cells were harvested.

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cells were collected by centrifugation, exposed to 1 % (w/v) tri-sodiumcitrate-2-hydrate hypotonie solution (for swelling) and fixed in glacial acetic acid/methanol, 1 +3. Drops of concentrated cell suspension were placed on slides, which were allowed to air-dry before being stained with 10% (v/v) Giemsa and mounted with Eukitt. For assessment of cell cycle kinetics, the slides were stained using a modified fluorescence-plus-Giemsa technique (FPG-technique). The slides were stained for 15 minutes with Hoechst 33258 (final concentration 4.5 µg/mL, rinsed and covered with phosphate butter (pH 6.9) during irradiation with ultra-violet light (e.g. Heraeus Sterisol®, 30 watt). After 90 min incubation in 12 x SSC at 60°C the slides were stained with 7% (v/v) Giemsa for ca. 10min, then rinsed with water and air-dried. After drying, the slides were mounted with Eukitt. On the basis of a "BUdR-control-culture", which was harvested after 2 days incubation with BUdR, the success of the differential staining was demonstrated in each instance.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): For the analysis of chromosomal aberrations, 100 cells per replicate culture were scored if possible, i.e. 200 cells per concentration level, except for the positive control in which only 100 cells were scored to prove the positive response. The number of chromosomes per metaphase was determined on the television monitor. Only metaphases with 2 n = 46 chromosomes were included in the analysis.

- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Using the vernier scale at the microscope stage, the coordinates of all metaphase spreads with structural aberrations were recorded.
All metaphase spreads were examined for both chromatid and chromosome aberrations, notably achromatic lesions (gaps), breaks, acentric fragments, deletions and exchange figures, with the following exception: an achromatic lesion (AL) may show a non-staining region greater than the diameter of the chromatid if there is no dislocation of the apparently detached part.
The incidence of cells with numerical alterations in chromosome number (excl. aneuploidy) such as endoreduplication and polyploidy was also recorded.
Endoreduplicated cells (endopolyploidy) were classified as quasitetraploid metaphases exhibiting chromosome pairing, whereas a polyploid cell is any metaphase containing multiple copies of the haploid number (n) of the chromosome complement, e.g. 3 n, 4 n etc.
All structural chromosome aberrations were assigned artificial lesion (or break) scores as folIows:
chromatid and chromosome (isochromatid) breaks, acentric fragments and deletions were scored as one lesion each exchanges, ringsand dicentrics were designated 2 lesions each.
The clastogenic potential of the compound was evaluated by calculating the breakage rate and the percentage of aberrant cells excluding ALs. The aberration frequency (% cells showing breaks excluding cells with ALs) in untreated or solvent-treated human
peripheral Iymphocyte cultures should be in the range of 0 - 3%. A break incidence of up
to 3% is therefore classified as a negative response, especially if a dose-dependent increase does not exist.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI); other: cell cycle progression
- Any supplementary information relevant to cytotoxicity: For determination of the cell cycle progression, 100 metaphase spreads per culture, treated with BUdR and stained with the FPG-technique, were analyzed for the occurrence of first, second and third mitosis in order to demonstrate that the first harvesting time chosen corresponds to approximately one to one and a half cell cycles after start of treatment.

Evaluation criteria:

please refer to any other information on materials and methods

Statistics:

The positive control group and the dose groups were compared to the appropriate negative control using Fisher's exact test, each at the level of significance α = 0.05.

Key result

Species / strain:

lymphocytes: human primary cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 75 µg/ml onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Evaluation of the data does not indicate any clastogenic potential of ZK 5690 in human peripheral blood Iymphocytes in vitro without or with an extrinsic metabolizing system. In both
assays ZK 5690 was tested up to concentration levels at which visible precipitates of the test compound occurred.
Therefore, ZK 5690 has to be considered as non-mutagenic in the chromosomal aberration assay on cultured human peripheral blood Iymphocytes under the experimental conditions described.

Executive summary:

ZK 5690 was examined for clastogenic activity in human lymphocytes from the peripheral blood (whole blood cultures treated ca. 48 hours after stimulation with phytohaemagglutinin).

The studies, which were conducted in the absence and presence of an extrinsic metabolizing system derived from phenobarbital and ß-naphthoflavone-induced male rat liver (89 mix), involved a range of ZK 5690 concentrations from 5 to 125 IJglml. As a rule the highest concentration tested should either reduce the mitotic index by approximately 50% or correspond to the substance's solubility limit, but not exceed 10-2 mol/l or 5 mg/ml . In the present study the solubility limit of ZK 5690 was the dose limiting factor as indicated by visible precipitations at the highest concentration scored for chromosomal aberrations. Generally, no distinct reduction of the mitotic index could be observed at any concentration tested. The following concentrations in culture were selected for the analysis of chromosomal aberrations (200 metaphases per concentration, i.e. 100 per duplicate culture):
10, 100 and 125 µg/ml in the assay without S9 mix (first harvesting time, 19 hours after a 4-hour treatment), whereby the mitotic index was reduced by 33% at the highest concentration.
125 µg/ml in the assay without S9 mix (second harvesting time, 40 hours after a 4-hour treatment).
10,75 and 125 µg/ml in the assay with S9 mix (harvesting time, 19 hours after a 4-hour treatment).
In none of the assays without and with S9 mix could a statistically significant increase in the frequency of structural chromosomal aberrations in human lymphocytes be observed in the blood cultures treated with 10-125 µg ZK 5690/ml. Positive controls with known mutagens (-S9 mix: triaziquone; +S9 mix: cyclophosphamide) produced the expected clastogenic effects.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Mar to May 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

21 July 1997

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not specified

Species / strain / cell type:

lymphocytes: human primary cells

Details on mammalian cell type (if applicable):

- Type and identity of media: MCCoy's 5a medium
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver homogenate
Type and composition of metabolic activation system:
S9 (batch no. 110997), derived from male Wistar rats pretreated with phenobarbital and ß-naphthoflavone, protein content 26.8 mg/mi, was prepared by RCC-CCR (Cytotest Cell Research, Roßdorf, Germany). The components of the standard 89 mix were 12.2% (viv) 89, 4 mmol/l NADP, 5 mmol/l glucose-6-phosphate, 8 mmol/I MgCI2, 34.3 mmol/l KCI and 50 mmol/l sodium phosphate buffer, pH 7.4.

Test concentrations with justification for top dose:

Assay without S9 mix:
1st harvesting: 1, 2.5, 5, 7.5, 10, 25, 50, 75, 100, 125 µg/ml ; 2nd harvsting: 50, 75, 100, and 125 µg/ml
Assay with S9 mix:
5, 10, 25, 50, 75, 100 and 125 µg/ml (first harvesting time) and 75, 100 and 125 µg/ml (second harvesting time).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solubility of ZK 5690 was tested in DMSO, yielding a solubility of > 12.5 mg/ml. But when this stock solution was added at 1% to tissue culture medium containing 15% (vIv) FCS precipitates of the test compound were visible starting at ca. 50 µg/ml.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: -S9 mix: triaziquone

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: One

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48 h
- Exposure duration/duration of treatment: without S9 mix: 21h first harvesting, 44 h second harvesting; with S9 mix: 24 h first harvesting, 44h second harvesting


FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure. To arrest the cells in the metaphase, colcemide (final concentration 0.08 µg/mL) was added ca. 3.5 h before the cells were harvested.

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): The cells were collected by centrifugation, exposed to 1 % (w/v) tri-sodiumcitrate-2-hydrate hypotonie solution (for swelling) and fixed in glacial acetic acid/methanol, 1 +3. Drops of concentrated cell suspension were placed on slides, which were allowed to air-dry before being stained with 10% (v/v) Giemsa and mounted with Eukitt. For assessment of cell cycle kinetics, the slides were stained using a modified fluorescence-plus-Giemsa technique (FPG-technique). The slides were stained for 15 minutes with Hoechst 33258 (final concentration 4.5 µg/mL, rinsed and covered with phosphate butter (pH 6.9) during irradiation with ultra-violet light (e.g. Heraeus Sterisol®, 30 watt). After 90 min incubation in 12 x SSC at 60°C the slides were stained with 7% (v/v) Giemsa for ca. 10min, then rinsed with water and air-dried. After drying, the slides were mounted with Eukitt. On the basis of a "BUdR-control-culture", which was harvested after 2 days incubation with BUdR, the success of the differential staining was demonstrated in each instance.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): For the analysis of chromosomal aberrations, 100 cells per replicate culture were scored if possible, i.e. 200 cells per concentration level, except for the positive control in which only 100 cells were scored to prove the positive response. The number of chromosomes per metaphase was determined on the
television monitor. Only metaphases with 2 n = 46 chromosomes were included in the analysis.

- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Using the vernier scale at the microscope stage, the coordinates of all metaphase spreads with structural aberrations were recorded.
All metaphase spreads were examined for both chromatid and chromosome aberrations, notably achromatic lesions (gaps), breaks, acentric fragments, deletions and exchange figures, with the following exception: an achromatic lesion (AL) may show a non-staining region greater than the diameter of the chromatid if there is no dislocation of the apparently detached part.
The incidence of cells with numerical alterations in chromosome number (excl. aneuploidy) such as endoreduplication and polyploidy was also recorded.
Endoreduplicated cells (endopolyploidy) were classified as quasitetraploid metaphases
exhibiting chromosome pairing, whereas a polyploid cell is any metaphase containing multiple copies of the haploid number (n) of the chromosome complement, e.g. 3 n, 4 n etc.
All structural chromosome aberrations were assigned artificial lesion (or break) scores as folIows:
chromatid and chromosome (isochromatid) breaks, acentric fragments and deletions were scored as one lesion each
exchanges, ringsand dicentrics were designated 2 lesions each.
The clastogenic potential of the compound was evaluated by calculating the breakage rate and the percentage of aberrant cells excluding ALs. The aberration frequency (% cells showing breaks excluding cells with ALs) in untreated or solvent-treated human peripheral Iymphocyte cultures should be in the range of 0 - 3%. A break incidence of up to 3% is therefore classified as a negative response, especially if a dose-dependent increase does not exist.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: mitotic index (MI); other: cell cycle progression
- Any supplementary information relevant to cytotoxicity: For determination of the cell cycle progression, 100 metaphase spreads per culture, treated with BUdR and stained with the FPG-technique, were analyzed for the occurrence of first, second and third mitosis in order to demonstrate that the first harvesting time chosen corresponds to approximately one to one and a half cell cycles after start of treatment.

Evaluation criteria:

please refer to any other information on materials and methods incl. tables

Statistics:

The positive control group and the dose groups were compared to the appropriate negative control using Fisher's exact test, each at the level of significance α = 0.05.

Key result

Species / strain:

lymphocytes: human primary cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 75 µg/ml onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

In conclusion, ZK 5690 showed no biologically relevant clastogenic potential in the chromosomal aberration assay with cultured human Iymphocytes from the peripheral blood whether in the absence or in the presence of rat liver S9 mix when investigated up to a recommended maximum concentration of 125 IJg/ml at which precipitates of the test compound could be observed. Therefore, ZK 5690 has to be considered as non-mutagenic in the chromosomal aberration assay on cultured human peripheral blood Iymphocytes under the experimental conditions described.

Executive summary:

There was no increase in the percentage of aberrant cells in the cultures treated with ZK 5690 at final concentrations of 10, 75 and 125 µg/ml (first harvesting time) and 125 µg/ml (second harvesting time) as compared with the solvent control. Additionally, there was no relevant reduction of the mitotic index at any concentration scored for chromosomal aberrations but visible precipitates of the test compound occurred from 75 µg/ml onwards.
Since the results were obviously negative in this case a statistical analysis was not performed.
Cyclophosphamide, the positive control, proved to be clearly clastogenic (p < 0.05).

Number of polyploid (including endoreduplicated) cells per concentration level observed in the course of scoring 200 metaphases for structural chromosomal aberrations:
First harvesting time
1/solvent; 0/10, 0/75 and 0/125 polyploid cells/µg ZK 5690/ml
Second harvesting time
0/solvent; 0/125 polyploid cells/µg ZK 5690/ml
The observed polypoidy rates do not arouse any suspicion of an aneugenic potential of the test compound.

In conclusion, ZK 5690 showed no biologically relevant clastogenic potential in the chromosomal aberration assay with cultured human lymphocytes from the peripheral blood whether in the absence or in the presence of rat liver S9 mix when investigated up to a recommended maximum concentration of 125µg/ml at which precipitates of the test compound could be observed. Therefore, ZK 5690 has to be considered as non-mutagenic in the chromosomal aberration assay on cultured human peripheral blood lymphocytes under the experimental conditions described.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1992

Deviations:

yes

Remarks:

no strain included to detect cross-linking (according to current version of the OECD TG 471, 2020)

Principles of method if other than guideline:

Preincubation modification was performed

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene locus

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate (S9 mix)

Test concentrations with justification for top dose:

All strains: 0.1, 0.25, 0.5, 1.0, 2.5, 5.0 mg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, phosphate buffer

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

N-dimethylnitrosamine

benzo(a)pyrene

cyclophosphamide

other: Anthracen-2-amine, 4-Nitro-o-phenylenediamine (4-NPDA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): E-06 dilution
- Test substance added: preincubation

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition

Evaluation criteria:

The plates were scored for the number of mutant colonies with an automated colony counter (Artek M 982B, Artek Systems Corporation, Farmingdale, NY, USA). In exceptional cases where reliable automatic counting is not possible, e. g. due to distinct precipitates of the test compounds,the colonies scored manually. The arithmetic means of the number of mutant colonies of the 3 parallel plates in the negative control groups were compared with those of the compound groups. A positive response was considered if at least 5 mg/plate or up to a toxic dose had been tested (ar the compound formed
precipitates in the agar) and if the number of induced revertants compared to the number of spontaneaus ones was higher than 2-fold. A dose-dependent increase in the number of revertants was also considered to indicate a mutagenic effect. A toxic effect of the substance on the background lawn of non-revertant bacteria and precipitates in the agar were examined stereomicroscopically.

Key result

Species / strain:

other: TA 1535, TA 1578, TA 1538, TA 98, TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

Evaluation of the data does not indicate that ZK 5690, is a mutagen in the Ames Salmonella/ microsome test using the pre-incubation modification, when tested up to the highest recommended dose level of 5.0 mg/plate.
This is in agreement with the results of an earlier study using the direct plate incorporation procedure (Schering report AN51) .

Executive summary:

ZK 5690, was examined for mutagenic activity in five histidine-dependent strains of Salmonella typhimurium (TA 1535 and TA 100 for detection of base-pair substitutions; TA 1537, TA 1538 and TA98 for detection of frame-shift mutations) using the modified Ames test (pre-incubation for 60 min at 37°C). The studies, which were conducted in the absence and presence of an extrinsic metabolizing system derived from Aroclor 1254-induced rat liver (S9 mix), employed a range of ZK 5690 doses from 0.1 to 5.0 mg per plate.
Negative controls and positive controls with known mutagens (anthracen-2-amine; benzo[a]pyrene; cyclophosphamide; 2-nitro-9H-fluorene; 4-nitro-o-phenylenediamine; sodium azide; N-nitrosodimethylamine) produced the expected numbers of revertant colonies.

None of the five tester strains showed increased reversion to prototrophy with ZK 5690, at the concentrations tested, either in the absence or presence of S9 mix.

Generally , precipitates in the agar were found starting at 1.0 mg/plate in the absence and presence of S9 mix. Growth inhibition of the background lawn was not observed.

 

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July - Nov 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1992

Deviations:

no

Principles of method if other than guideline:

Direct plate incorporation procedure was performed.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

liver S9-mix from Aroclor 1254 -treated rats

Test concentrations with justification for top dose:

seven concentrations from 10 to 5000 µg/plate
(10, 33.3, 100, 333.3, 1000, 2500, 5000 µg/ plate)

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and phosphate buffer pH 7.4, 0.1 mol/l

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine; 2-Aminoanthracene

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

Evaluation criteria:

The plates were scored for the number of mutant colonies with an automated colony counter (Artek Systems Corporation, BIOSYS GmbH, D-61184 Karben; F.R.G.). The background growth of the bacteria was judged visuallyon a light bench. Whenever precipitation ofthe test article occurred, the colonies were counted manually.

The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates

A test article is considered positive if either a dose related increase in the number of revertants or a biological relevant increase for at least one test concentration is induced. A test article producing neither a dose related increase in the number of revertants nor a biological relevant positive response at any one of the test points is considered nonmutagenic in this system.
A significant response is described as folIows:
A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 100 and TA 102 or thrice on TA 1535, TA 1537, and TA 98.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

No increase of reverse gene mutations in bacteria induced by the test item up to maximum concentration of 0.5 mg/ plate.

Executive summary:

In the Ames test no toxic effeets, evidenced by a reduction in the number of revertants, occurred in the test groups with 1,2-Methylen-4,6-dienacetat with and without metabolic activation.
No substantial increases in revertant colony numbers of any of the five tester strains (TA 1535, TA 1537, TA 98, TA 100, TA 102) were observed following treatment with ZK 5.690 at any concentration level, either in the presence or absence of metabolic activation (S9 mix). In strain 1537 the numbers of revertants were slightly above the corresponding solvent control in an test points with metabolic activation. This effect does not indicate a true mutagenic effect since the actual numbers of revertant colonies are low and well within the range of historical negative control data. There was also no tendency of high er mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate control mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In the bacterial reverse mutation assay (OECD TG 471) no adverse effects neither with the plate incorporation nor with the pre-incubation  method was observed for the test item.

In addition, 2 independent experiments equivalent to OECD TG 473 (Chromosomal abberation) in human peripheral lymphocytes showed no clastogenic effect.

Endpoint Conclusion: No adverse effect on genotoxicity observed (negative)

Justification for classification or non-classification

Based on the results there is no classification required according to Directive 67/548/EEC and Regulation (EC) 1272/2008 (CLP).