4,4'-[(6-chloro-1,3,5-triazine-2,4-diyl)diimino]bis[5-hydroxy-6-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonic] acid, sodium salt

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:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

October 1991 to January 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to OECD test guidelines and in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Structural Analogue 02

IUPAC Name:

Structural Analogue 02

Details on test material:

see below

Method

Target gene:

Not applicable.

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocyte

Details on mammalian cell type (if applicable):

Human peripheral blood was obtained by venepuncture on the days of culture initiation from two healthy non-smoking human donors, Donor 1 being male, and Donor 2 being female. Both donors have a previously established low incidence of chromosomal damage in their peripheral blood lymphocytes.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

140 to 6990 μg/ml for cultures treated in the absence of S9-mix
700 to 6990 μg/ml for cultures treated in the presence of S9-mix

Vehicle / solvent:

Dosing solutions of the test substance were prepared in RPMI-1640 culture medium and serial dilutions in culture medium were carried out as required. Mitomycin C and cyclophosphamide were prepared in sterile double deionised water, CTL reference number YO4517/01O.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Blank medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: x2 +ve control substances

Remarks:

+ve control substances detailed below

Details on test system and experimental conditions:

Summary of Methodology: Aliquots of the test substance solutions, the positive control solutions and S9-mix were administered to the cultures, as appropriate to the test being conducted, approximately 48 hours after culture initiation.

Cultures treated in the presence of S9-mix were then incubated at 37°C for approximately 3 hours, after which the growth medium was replaced with fresh medium, and the cultures were re-incubated at 37°C for the remainder of the 72 hour growth period. Cultures treated in the absence of S9-mix were incubated at 37°C throughout the remainder of the 72 hour growth period.

Dividing lymphocytes were arrested in metaphase by treatment with colcemid (at a final concentration of 0.4ug/ml) 70 or 94 hours after culture initiation for a period of 2 hours at 37°C. At 72 or 96 hours the cells were harvested by low speed centrifugation (400g for S minutes) and then subjected to hypotonic treatment for approximately 10 minutes in 0.07SM potassium chloride (KCl) solution at room temperature followed by fixation in methanol/glacial acetic acid. Single drops of cell suspension were dropped onto clean, moist slides which were then stained in Giemsa and mounted in DPX.

Mitotic index was assessed by examining 1000 lymphocytes per culture and calculating the percentage of cells in metaphase. For each donor, both in the presence and absence of S9-mix, cultures treated with Substance H112339 at three concentrations were selected for chromosomal aberration analysis at the 72 hour sampling time. For cultures treated in the presence of S9-mix, the highest level was equivalent to the limit dose for the assay. For cultures treated in the absence of S9-mix., the highest level was limited by residual test material on the slides which obscured the metaphase cells. In addition, cultures from the male donor treated with Substance H112339 at 1440 and 6990ug/ml, were selected for analysis at the 96 hour sampling time, in the absence and presence of S9-mix respectively.

The slides were coded prior to analysis and one hundred cells in metaphase were analysed from each selected culture for the incidence of structural chromosomal damage, according to the criteria recommended by Scott et al (1990). If under high power magnification the quality of the metaphase precluded accurate scoring (eg excessive fuzziness or overlapping chromosomes) the cell was not included in the analysis.

Evaluation criteria:

The total number of abnormal cells, excluding those with only gap-type aberrations, for each of the treatment groups was compared to the appropriate medium control values using the Fisher's Exact Test (one sided),

Statistics:

The total number of abnormal cells, excluding those with only gap-type aberrations, for each of the treatment groups was compared to the appropriate medium control values using the Fisher's Exact Test (one sided)

Results and discussion

Test results

Species / strain:

lymphocytes: human peripheral blood lymphocyte

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Determination of Mitotic Indices and Selection of Dose Levels

The recorded and mean mitotic indices for the medium controls and the test substance treated cultures, in the absence or presence of S9-mix, from the main cytogenetic tests.

Two independent main cytogenetic tests were carried out using up to seven concentrations ranging from 140 to 6990 μg/ml in the absence of S9-mix and a range of four concentrations from 700 to 6990 μg/ml in the presence of S9-mix.

Residual test material after fixation was noted in cultures treated with the test substance at all concentrations tested. This ranged from slight pink colouration of the cell pellet to visible particles of the test material on the slides. This precipitation on the slides limited the concentration of the test substance, in the absence of S9-mix, which could be selected for chromosomal aberration analysis.

At the 72 hour sampling time no significant reductions in mitotic activity, compared to the medium control values, were observed in the cultures from either donar treated in the presence of S9-ntix. Reductions in mitotic activity, compared to the medium control values, of 28% for the male donor and 34% for the female donor were observed in cultures treated with the test substance at 3500 μg/ml in the absence of S9-mix and harvested at the 72 hour sampling time.

From the results of these tests, cultures treated with the test substance at concentrations of 350, 1400 and 3500 μg/ml and 700, 3500 and 6990 μg/ml in both donors, in the absence and presence of S9-mix respectively, were selected for chromosomal aberration analysis at the 72 hour sampling time-

At the 96 hour sampling time significant reductions in mitotic activity of 69% were observed in cultures treated with the test substance at the highest concentrations selected for chromosomal aberration analysis in the presence and absence of S9-mix.

Cultures from the male donor treated with the test substance at 1440 and 6990μg/ml, in the absence and presence of S9-mix respectively, were selected for analysis at the 96 hour sampling time.

Results of Chromosomal Aberration Analyses

One hundred metaphases were scored for each culture. No statistically or biologically significant increases in chromosomal aberration frequencies (including or excluding gap-type aberrations) were observed in cultures from either donor, in the presence or absence of auxiliary metabolic activation at the 72 hour sampling time or in any of the cultures at the 96 hour sampling time.

The test system was shown to be sensitive to direct acting clastogens from the responses obtained with mitomycin C, and sensitive in detecting clastogens which require metabolic activation as demonstrated by the responses observed with cyclophosphamide.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

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

Under the conditions of test, the test substance isconsidered not to be clastogenic to human lymphocytes in vitro.

Executive summary:

The test material was assessed in an in vitro cytogenetic assay in cultured human peripheral blood lymphocytes to determine if the test material had any clastogenic activity. The procedures and experimental design employed complied with the recommendations of the relevant OECD Guideline 473 (1983) and the UKEMS Recommended Procedures for Basic Mutagenicity Tests (Kirkland, 1990). The test substance was tested over a range of concentrations, both in the presence and absence of S9-mix, using blood from 2 donors. In the presence of S9-mix the highest concentration of the test substance selected for chromosomal aberration analysis in this assay was equivalent to the limit dose of the assay whereas in the absence of S9-mix, the highest concentration selected for chromosomal aberration analysis was limited by residual test material on the slides.

No statistically or biologically significant increases in chromosomal aberration frequencies, compared to the medium control values, were observed in any cultures treated with the test substance, in the presence or absence of S9-mix, at either sampling time.

The sensitivity of the test system, and the metabolic activity of the S9-mix employed, were clearly demonstrated by the increases in the frequencies of chromosomal aberrations induced by the positive control agents, mitomycin C and cyclophosphamide.

The data obtained in this study therefore show that the test substance did not induce chromosomal damage in human peripheral blood lymphocytes following in vitro treatment in the presence or absence of S9-mix.

Under the conditions of test, the test substance isconsidered not to be clastogenic to human lymphocytes in vitro.