Reaction mass of 3,6-Bis(3-chlorophenyl)-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione, 3-(3-Chlorophenyl)-6-(4-chlorophenyl)-2,5-dihydro- pyrrolo[3,4-c]pyrrole-1,4-dione and 3,6-Bis(4-chlorophenyl)-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidelines stipulated by the Japanese Ministry of Labor and Japanese Ministry of International Trade and Industry

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Details on test material:

- Description: Orange-red powder
- Test substance storage: At room temperature in the dark
- Stability under storage conditions: Stable
- Expiry date: 15 August 2007

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and ß-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

Dose range finding study:
3 hours exposure time: 0.1, 0.3, 1, 3 and 10 µg/mL, with and without S9-mix.
24 h and 48 h exposure time: 0.1, 0.3, 1, 3, 10 and 33 µg/mL, without S9-mix.

First cytogenetic assay:
3 hours exposure time: 1, 3 and 10 µg/mL, With and without S9-mix.

Second cytogenetic assay:
24 h and 48 h exposure time: 1, 3, 10 and 33 µg/mL, Without S9-mix.
3 h exposure time: 1, 3 and 10 µg/mL, With S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide
- Justification for choice of solvent/vehicle: the test substance was insoluble in water; in DMSO a homogeneous suspension was obtained

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DOSE RANGE FINDING:
In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. The test substance was tested in the absence and in the presence of 1.8% (v/v) S9-fraction. Lymphocyte cultures (0.4 mL blood of a healthy male donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed to selected doses of the test substance for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. The highest tested concentration was determined by the solubility of the test substance in the culture medium at the 3 h exposure time. At the 24 and 48 h exposure time, the test substance was tested beyond the limit of solubility to obtain adequate toxicity data. After 3 h exposure to the test substance in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and cells were rinsed with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 mL culture medium and incubated for another 20-22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h and 48 h fixation time).

MAIN TEST
- First cytogenetic assay: Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of the test substance for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and the cells were rinsed once with 5 mL HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 mL culture medium and incubated for another 20-22 h (24 h fixation time).
- Second cytogenetic assay: Lymphocyte cultures were cultured for 48 h and thereafter exposed in duplicate to selected doses of the test substance for 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. After 3 h exposure, the cells exposed to the test substance in the presence of S9-mix were separated from the exposure medium by centrifugation (5 min, 150 g). The supernatant was removed and the cells were rinsed once with 5 mL of HBSS and incubated in 5 mL culture medium for another 44-46 h (48 h fixation time). The cells that were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time).
- Chromosome preparation: During the last 2.5 to 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/mL medium) (Acros Organics, Belgium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (150 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v).
- Preparation of slides: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the NOTOX study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10-30 min with 5% (v/v) Giemsa (Merck) solution in tap water. Thereafter slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene (Klinipath, Duiven, The Netherlands) before they were embedded in MicroMount (Klinipath) and mounted with a coverslip.
- Analysis of slides for chromosome aberrations: To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated.

NUMBER OF REPLICATIONS: duplicate in two independent experiments.

DETERMINATION OF CYTOTOXICITY:
The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed of those cultures with an inhibition of the mitotic index of about 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations. In case the test substance was not cytotoxic and/or difficult to dissolve in aqueous solutions, the highest concentration analysed at the 3 h exposure time was determined by the solubility in the culture medium. If dose related cytotoxicity was observed, the highest concentration analysed at the 24 and 48 h continuous exposure times was based on toxicity irrespective of the solubility of the test substance in the culture medium. However, the extent of precipitation may not interfere with the scoring of chromosome aberrations.

Evaluation criteria:

A chromosome aberration test was considered acceptable if it met the following criteria:
- The number of chromosome aberrations found in the solvent control cultures should reasonably be within the laboratory historical control data range.
- The positive control substances should produce a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
- A homogeneous response between the replicate cultures is observed.
- A possible precipitate present on the slides should not interfere with the scoring of chromosome aberrations.
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
- It induced a dose-related statistically significant (Chi-square test, two-sided, P < 0.05) increase in the number of cells with chromosome aberrations.
- A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, two-sided, P < 0.05) increase in the number of cells with chromosome aberrations.

The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.

If P (two-tailed) is small (P< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence level.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each exposure group was compared to that of the solvent control using Chi-square statistics.

If P (two-tailed) is small (P< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95% confidence level.

Results and discussion

Test results

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
At a concentration of 10 µg/mL the test substance precipitated in the culture medium.

RESULTS ON GENOTOXICITY
- First cytogenetic assay: In the absence of S9-mix the test substance induced a statistically significant increase in the number of cells with chromosome aberrations at the lowest tested concentration only, when gaps were included. Since the type of aberrations observed were only breaks and gaps, the increase was not dose related and moreover the number of cells with chromosome aberrations was well within our historical control data range, the increase was considered not biologically relevant. In the presence of S9-mix, the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.
- Second cytogenetic assay: Both in the absence and presence of S9-mix the test substance did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations. No effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.
- The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

The substance does not cause chromosome aberrations in cultivated mammalian cells.