Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis([1,1'-biphenyl]-4-yl)-2,5-dihydro-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames test: according to OECD TG 471, GLP, S. typhimurium/ E.coli, with and without metabolic activation, negative

- Mouse lymphoma assay (MLA): according to OECD TG 476, GLP, with and without metabolic activation, negative

- Chromosome aberration (CA) test: according to OECD TG 473, GLP, V79 cells, with and without metabolic activation, negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992

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:

adopted May 26, 1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Substance type: Organic
- Physical state: Solid
- Lot/batch No.: Z-2281/1,2,4,5
- Expiration date of the lot/batch: April 01, 1997
- Storage condition of test material: at room temperature in the dark

Target gene:

His: Salmonella; Trp: E. Coli

Species / strain / cell type:

E. coli WP2

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

10; 100; 333.3; 1000; and 5000 µg/plate

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see: "Details on test system and conditions"

Details on test system and experimental conditions:

POSITIVE CONTROLS:
Without metabolic activation (-S9-mix):
- TA1535, TA 100: sodium azide (SA), 10 µg
- TA1537, TA 98: 4-nitro-o-phenylene-diamine, 4-NOPD 50 µg
- WP2uvrA, WP2 : methylmethanesulfonate (MMS), 10 µl

With metabolic activation (+S9-mix):
- TA1537, : 2-aminoanthracene (2AA), 2.5 µg
- TA1535, TA 1537, TA98 and TA100: 2-aminoanthracene (2AA), 2 µg
- WP2uvrA and WP2: 2-aminoanthracene (2AA), 2 µg

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Incubation period: 48 hours. After this period revertant colonies (histidine independent for Salmonella typhimurium bacteria and tryptophan independent for Escherichia coli were counted.
- COLONY COUNTING:
- Exposure duration: The revertant colonies (histidine independent/ tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

NUMBER OF REPLICATIONS: 3

Evaluation criteria:

A test article is considered as mutagenic if in the strains TA 100, WP2, and its uvrA derivate the number of reversions will be at least twice as high and in the strains TA 1535, TA 1537, and TA 98 it will be at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent increase in the number of revertants isregarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate without S9 mix in experiment I

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRECIPITATE: Not mentioned.

TOXICITY: Toxic effects evidenced by a reduction in revertant colony numbers occurred only in strain TA 1535 at 5000 µg/plate without S9 mix in experiment I. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

MUTAGENICITY: No increase in the number of revertants was observed upon treatment with the test substance under all conditions tested. All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.

Table 1: Number of revertants in the control or after treatment with the test substance

 

First experiment (10 - 5000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 1535	no	15	1.1	no	negative
	yes	15	1.2	no	negative
TA 1537	no	6	1.1	no	negative
	yes	6	1.4	no	negative
TA 98	no	20	1.2	no	negative
	yes	33	1.2	no	negative
TA 100	no	78	0.9	no	negative
	yes	88	1.1	no	negative
E. coli WP2 uvrA	no	38	1.2	no	negative
	yes	41	1.1	no	negative
E. coli WP2	no	30	1.6	no	negative
	yes	40	1.2	no	negative
Second experiment (10 - 1000 µg/plate)
Strain	Metabolic activation system	mean revertants in Controls	maximum revertant factor	dose dependency	Assessment
TA 1535	no	14	1.1	no	negative
	yes	15	1.2	no	negative
TA 1537	no	11	1.2	no	negative
	yes	15	1.2	no	negative
TA 98	no	24	1.2	no	negative
	yes	41	1.3	no	negative
TA 100	no	90	1.1	no	negative
	yes	91	1.0	no	negative
E. coli WP2 uvrA	no	39	1.4	no	negative
	yes	47	1.1	no	negative
E. coli WP2	no	57	1.1	no	negative
	yes	56	1.1	no	negative

Conclusions:

Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

In a GLP conform study according to OECD guideline 471, the potential of the test substance to induce gene mutations was investigated in two independent experiments using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA and WP2.

In both experiments, the test substance was tested up to a concentration of 5000 µg/plate in the absence and presence of S9-mix. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed. The test substance did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strains WP2uvrA and WP2, both in the absence and presence of S9-metabolic activation. These results were confirmed in two independent experiments. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-1993

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

- Substance type: Organic
- Physical state: Solid
- Lot/batch No.: Z-2281/1,2,4,5
- Expiration date of the lot/batch: April 01, 1997
- Storage condition of test material: at room temperature in the dark

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: LMP, Technical University Darmstadt, D-6100 Darmstadt
- Suitability of cells: The V79 cell line has been used for many years in in vitro experiments with success.

For cell lines:
- Number of passages if applicable:
- Methods for maintenance in cell culture: The cells were subcultured twice weekly.
- Doubling time: 12 - 16 h in stock cultures

- Periodically checked for karyotype stability and the absence of mycoplasma contamination: yes, before storing (freezing), each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remained similar because of the reproducible characteristics of the cells.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: MEM (minimal essential medium) supplemented with 10 % fetal calf serum (FCS). The cell cultures were incubated at 37''C and 4.5 % carbon dioxide atmosphere.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9-mix

Test concentrations with justification for top dose:

- 18 h: 0.1; 0.3; 0.6; 1.0; 3.0; 6.0 µg/ml (with and without S9 mix);
- 28 h: 0.6; 1.0; 3.0; 6.0 µg/ml with and without S9 mix)

Only the following concentrations were evaluated for for chromosome aberrations:
- 18 h: 0.6; 3.0; 6.0 (with and without S9 mix)
- 28 h: 6.0 µg/L (with and without S9 mix)

Vehicle / solvent:

Dimethyl sulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: The cells were seeded into Quadriperm dishes which contained microscopic slides (2 chambers per dish and test group). In each chamber 5 x 10^4 - 1 x 10^5 cells were seeded with regard to preparation time.
- Test substance added in medium: On the day of the experiment (immediately before treatment), the test article was dissolved in DMSO. The test article was suspended in DMSO, heated to 160°C in a gastight vessel and stirred for 20 minutes. The concentration of the stock solution was 1 mg/ml. The solvent was chosen according to its solubility properties and its non-toxcicity for the cells. The final concentration of DMSO in the culture medium did not exceed 1 % v/v.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 48h (28h preparation interval) and 55h (18h preparation interval)
- Exposure duration: 4h
- Fixation time (start of exposure up to fixation or harvest of cells): 18 and 28 hours
- Harvest time after the end of treatment (sampling/recovery times):

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colcemide (0.2 µ.g/ml medium); 2.5 h
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): 10-30 min with 5% (v/v) Giemsa solution in tap water
- Number of cells spread and analysed per concentration: At least 100 metaphase chromosomes per culture
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie", only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis Cytogenetik Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Plating efficiency (seeding of ca. 500 cells per flask), mitotic index (% cells in mitosis); number of polyploid cells

Rationale for test conditions:

The test article was suspended in DMSO. The test article precipitated strongly in culture medium and particles were microscopically visible at concentrations of 0.60 µg/ml and higher. Due to this strong precipitation cytogenetic evaluation of cultures was prevented at concentrations higher than 6.0 µg/ml.

Evaluation criteria:

ACCEPTABILITY OF ASSAY:
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
a) the number of aberrations found in the negative and/or solvent controls fall within the laboratory historical control data range: 0.00 % - 4.00 %.
b) the positive control substances should produce significant increases in the nvimber of cells with structural chromosome aberrations.

DATA EVALUATION AND STATISTICAL PROCEDURES:
A test article is classified as mutagenic if it induces reproducibly either a significant concentration-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points. A test article producing reproducibly neither a significant concentration-related increase in the number of structural chromosomal aberrations nor a significant and reproducibly positive response at any one of the test points is considered nonmutagenic in this system. This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: At a concentration of 0.6 µg/ml the test substance precipitated in the culture medium. Therefore, a concentration of 6 µg/ml was used as the highest concentration of the test substance.

STUDY RESULTS
- Concurrent vehicle negative and positive control data are given
- In both experiments, EMS (0.33 mg/ml) and CPA (2.8 ng/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements: In both experiments, at both fixation intervals (with and without S9 mix) the mitotic index was not reduced distinctly even at the highest concentration used (6.0 ng/ml).
- Genotoxicity results: In experiment I and II, at both fixation intervals and in the absence and presence of S9 mix the test article did not increase the frequency of cells with aberrations to a statistically and biologically relevant extent.
The aberration rates of the cells after treatment with the test article (exp.I: 0.00 % - 1.00 %; exp. II 0.00 % - 2.00%) were in the range of the negative and solvent control values: exp.I: 0.50 % - 1.50 %; exp. II: 0.00 % - 3.00 % and in the range of our historical control data: 0.00 % - 4.00 %.
- Occurence of polyploid metaphase: In both experiments, no biologically relevant deviations from the control data (exp. I: 0.5 % - 5.0 %; exp. II: 0.5 % - 3.5 %) were found after treatment with the test article (exp. I: 2.0 % - 4.5 %; exp. I I : 1 . 5 % - 4 . 0 % ).

Conclusions:

Under the experimental conditions of this studiy the test substance did not induce reproducibly structural chromosome aberrations in the V79 Chinese hamster cell line.

Executive summary:

The possible in vitro cytogenicity of the test substance was assessed in a GLP conform study according to OECD guideline 473 in two independent experiments using V79 cells. In each experimental group two parallel cultures were set up and cells were treated with the test substance formulated in DMSO for 4 hours in the absence and presence of metabolic activation. The preparation of chromosomes was done 18 h (0.6; 3.0; 6.0 µg/ml), and 28 h (6 µg/ml) after start of treatment. Per culture 100 metaphases were scored for structural chromosomal aberrations except for the positive control cultures (25 metaphases were scored).

The concentration range of the test article applied was limited by its solubility. The solubility of the test article in aqueous solutions (e.g. culture medium, aqua dest.) and other organic solvents (e.g. DMSO, acetone, ethanol) was very poor. Therefore, a suspension of the test article had to be prepared. The evaluation of cultures treated with concentrations higher than 6.0 µg/ml was prevented due to a very strong precipitation of the test article. There was no reproducible reduction of the mitotic indices at any fixation interval (with and without S9 mix) up to the highest concentration used. In both independent experiments, the test substance did not induce a statistically significant or biologically relevant increase in the frequency of cells with chromosome aberrations in the absence and in the presence of S9-mix at both fixation intervals. The aberration rates of the cells were in the range of the negative and solvent control values and in the range of our historical control data. There was no increase in the polypoloidy index.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011 - 2012

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:

21st July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

- Name: Cinilex DPP Rubine SR5H
- Other Identifiers: Pigment Red 264, C.I. 561300
- Batch/Lot: Standard Lot H11601; Production Lot D1254710P1
- Manufacturing Date: 2010/07/30

Information from Supplier, not contained in study report:
- Specific surface area (BET): 22.8 m^2/g (not a nanomaterial)
- Purity: 99.1%

Target gene:

thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

The liver extract is obtained from rats, which were pre-treated with phenobarbital/β-naphthoflavone.

Test concentrations with justification for top dose:

Assay 1:
3-hour treatment (±S9 Mix): 50; 100; 250; 500 and 1000 μg/mL
Assay 2:
24-hour treatment (-S9 Mix): 50; 100; 250; 500 and 1000 μg/mL
3-hour treatment (+S9 Mix): 50; 100; 250; 500 and 1000 μg/mL

The test item is insoluble and was investigated bevond its limit of solubility under culture conditions. In the examined concentration range noticeable cytotoxicity did not occur at the 3-hour and 24-hour treatments.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1x 10^7
- Test substance was added in RPMI 5 medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3h (Assay 1, with and without metabolic activation), 24h (assay 2, witout metabolic activation), 3h (assay 2, with metabolic activation)
- Harvest time after the end of treatment

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days
During the expression period, subculturing was performed daily with the aim of not exceeding 1x10^6 cells per mL and, where possible and retaining a total of at least 5x10^6 cells/flask. For this purpose cell densities were adjusted to a concentration of 2x10^5/mL and transferred to flasks for further growth. From observations on recovery and growth of the cultures during the expression period, six dose levels plus negative and positive controls were plated for viability and 5-trifluorothymidine (TFT) resistance.
- Fixation time (start of exposure up to fixation or harvest of cells): 16-17 days
- Method used: microwell plates
- Selective agent: itrifluorothymidine at 3 μg/mL applied for 2 weeks
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2x10^3 cells per well, identification by eye using background illumination
- Criteria for small (slow growing) and large (fast growing) colonies: not specified

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning efficiency

METHODS FOR MEASUREMENTS OF GENOTOXICITY: counting of large and small colonies

Evaluation criteria:

The test item is considered to be mutagenic in this assay if all the following criteria are met (based on Moore et al.):
1. The assay is valid;
2. Statistically significant (p < 0.05) increases in mutation frequency are observed in treated cultures compared to the corresponding vehicle control values at one or more concentrations;
3. The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
4. There is a significant dose-relationship as indicated by the adequate trend analysis;
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative control value [e.g.: If the vehicle control MF is 50 x 10-6, then one of the test cultures must have an MF of at least (50+126) x 10 -6 = 176 x 10 -6 to meet the GEF criterion for a positive call.].

The assay is considered valid if all of the following criteria are met (based on M. Moore et al.):
1. The mutant frequency in the negative (vehicle) control cultures fall within the normal range (above 50-170 mutants per 106 viable cells).
2. The positive control chemicals induce a statistically significant increase in the mutant frequency.
3. The plating efficiency (PE viability) of the negative controls is within the range of 65 % to 120 % on Day 3 (at the end of the expression period).
4. At least four test concentrations are present, where the highest concentration produces 80-90% toxicity, precipitation, or is 5 mg/mL, or the highest practical concentration.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

and also precipitation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Validity of the Mutation Assays
1. The spontaneous mutation frequency of the negative (vehicle) control cultures were within the expected normal range (50-170 mutants per 106 viable cells) in the performed experiments in line with the historical controls.
2. The positive control chemicals (Cyclophosphamide in the presence and 4-Nitroquinoline-N-oxide in the absence of S9 Mix) induced a statistically significant increase in the mutant frequency (2 Sample t-Test, α=0.01).
3. The plating efficiency (PEviability) of the RPMI5 Medium vehicle control was within the range of 65 % to 120 % in all cases.
4. The concentrations applied in the assays (at the 3-hour and 24-hour treatments) were chosen according to the solubility and cytotoxicity results of the pre-experiments. The test item was considered as relatively insoluble therefore it was investigated beyond its limit of solubility under culture conditions.

- There were no effects on pH or osmolality.

- For further information, please see the attached data in section "Attached background material" and "Illustration".

Conclusions:

Under the experimental conditions of this study, the test substance was found to be non mutagenic in mouse lymphoma cells.

Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus according to OECD guideline 476 an GLP, in order to assess the potential of the test substance to cause gene mutations and/or chromosome damage. In two independent experiments, treatments were carried out for 3 hours with and without metabolic activation (±S9 Mix) and for 24 hours without metabolic activation (-S9 Mix) at concentrations of 50; 100; 250; 500 and 1000 µg/mL. Based on the results of preliminary solubility and toxicity tests and regarding the practical difficulties with the test item handling, the test item was suspended and diluted in RPMI 5 Medium and the RPMI 5 Medium was parallel investigated as vehicle control. After the treatment periods, cell cultures were washed and transferred to flasks at a density of 2x 10⁵/mL for growth through the expression period (approximately 2 days). At the end of the expression period cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT). Thereafter, the relative harmonised survival, the relative total growth of the cells, the viability (colony-forming ability at the end of the 2-day expression period following the treatment) and the potential mutagenicity (5-trifluorothymidine resistance) were determined. Since the result of the first assay (treatment period of 3 hours only) was non mutagenic, the second assay was performed with the originally planned treatments based on the preliminary cytotoxicity test with treatment periods of 3 (in presence of metabolic activation) and 24 hours (in absence of metabolic activation). The assays fulfilled the validity criteria for the negative control and positive control treatments as well as the number of analyzable concentration levels (at least four). In the examined concentration range noticeable cytotoxicity did not occur at the 3-hour and 24-hour treatments. The obtained mutation frequencies (in absence and also in presence of exogenous metabolic activation) did not show dose-related tendencies, remained far below the relevant GEF thresholds for positive call and remained in the validity criterion range of the negative vehicle control cultures. Therefore, the test substance was found to be non mutagenic in mouse lymphoma cells under the experimental conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro:

Ames test:

In a GLP conform study according to OECD guideline 471, the potential of the test substance to induce gene mutations was investigated in two independent experiments using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA and WP2.

In both experiments, the test substance was tested up to a concentration of 5000 µg/plate in the absence and presence of S9-mix. The bacterial background lawn was not reduced at all concentrations tested and no decrease in the number of revertants was observed. The test substance did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strains WP2uvrA and WP2, both in the absence and presence of S9-metabolic activation. These results were confirmed in two independent experiments. Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

 

Cytogenicity in mammalian cells:

The possible in vitro cytogenicity of the test substance was assessed in a GLP conform study according to OECD guideline 473 in two independent experiments using V79 cells. In each experimental group two parallel cultures were set up and cells were treated with the test substance formulated in DMSO for 4 hours in the absence and presence of metabolic activation. The preparation of chromosomes was done 18 h (0.6; 3.0; 6.0 µg/ml), and 28 h (6 µg/ml) after start of treatment. Per culture 100 metaphases were scored for structural chromosomal aberrations except for the positive control cultures (25 metaphases were scored).

The concentration range of the test article applied was limited by its solubility. The solubility of the test article in aqueous solutions (e.g. culture medium, aqua dest.) and other organic solvents (e.g. DMSO, acetone, ethanol) was very poor. Therefore, a suspension of the test article had to be prepared. The evaluation of cultures treated with concentrations higher than 6.0 µg/ml was prevented due to a very strong precipitation of the test article. There was no reproducible reduction of the mitotic indices at any fixation interval (with and without S9 mix) up to the highest concentration used. In both independent experiments, the test substance did not induce a statistically significant or biologically relevant increase in the frequency of cells with chromosome aberrations in the absence and in the presence of S9-mix at both fixation intervals. The aberration rates of the cells were in the range of the negative and solvent control values and in the range of our historical control data. There was no increase in the polypoloidy index.

 

Mutagenicity in mammalian cells:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus according to OECD guideline 476 an GLP, in order to assess the potential of the test substance to cause gene mutations and/or chromosome damage. In two independent experiments, treatments were carried out for 3 hours with and without metabolic activation (±S9 Mix) and for 24 hours without metabolic activation (-S9 Mix) at concentrations of 50; 100; 250; 500 and 1000 µg/mL. Based on the results of preliminary solubility and toxicity tests and regarding the practical difficulties with the test item handling, the test item was suspended and diluted in RPMI 5 Medium and the RPMI 5 Medium was parallel investigated as vehicle control. After the treatment periods, cell cultures were washed and transferred to flasks at a density of 2x 10⁵/mL for growth through the expression period (approximately 2 days). At the end of the expression period cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT). Thereafter, the relative harmonised survival, the relative total growth of the cells, the viability (colony-forming ability at the end of the 2-day expression period following the treatment) and the potential mutagenicity (5-trifluorothymidine resistance) were determined. Since the result of the first assay (treatment period of 3 hours only) was non mutagenic, the second assay was performed with the originally planned treatments based on the preliminary cytotoxicity test with treatment periods of 3 (in presence of metabolic activation) and 24 hours (in absence of metabolic activation). The assays fulfilled the validity criteria for the negative control and positive control treatments as well as the number of analyzable concentration levels (at least four). In the examined concentration range noticeable cytotoxicity did not occur at the 3-hour and 24-hour treatments. The obtained mutation frequencies (in absence and also in presence of exogenous metabolic activation) did not show dose-related tendencies, remained far below the relevant GEF thresholds for positive call and remained in the validity criterion range of the negative vehicle control cultures. Therefore, the test substance was found to be non mutagenic in mouse lymphoma cells under the experimental conditions of this study.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008:

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008 as amended for the thirteenth time in Regulation (EC) No. 2018/1480.