1,7-Naphthalenedisulfonic acid, 4-amino-3-[(1E)-2-(2,4-disulfophenyl)diazenyl]-5-hydroxy-6-[(1E)-2-(4-nitro-2-sulfophenyl)diazenyl]-, sodium salt (1:5)

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

Study period:

01-05-2015 to 23-07-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Performed in accordance to OECD guideline and GLP compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

pentasodium 4-amino-3-[(1E)-2-(2,4-disulfonatophenyl)diazen-1-yl]-5-hydroxy-6-[(1E)-2-(4-nitro-2-sulfonatophenyl)diazen-1-yl]naphthalene-1,7-disulfonate

EC Number:

811-858-8

Cas Number:

2149571-68-4

Molecular formula:

C22 H11 N6 O18 S5 .5Na

IUPAC Name:

pentasodium 4-amino-3-[(1E)-2-(2,4-disulfonatophenyl)diazen-1-yl]-5-hydroxy-6-[(1E)-2-(4-nitro-2-sulfonatophenyl)diazen-1-yl]naphthalene-1,7-disulfonate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Method

Species / strain

Species / strain / cell type:

lymphocytes: Peripheral human lymphocytes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

512, 1600 and 5000 µg/ml culture medium

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water: 20-23%
- Justification for choice of solvent/vehicle: 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 in the solvent control cultures was within the laboratory historical control data range.

Controls

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration: 3, 24 and 48 hrs
- Expression time (cells in growth medium): 48 + or - 2 h
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells): first cytogenetic assay: 24 h, second cytogenetic assay: 24 h and 48 h

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/ml medium) (Acros Organics, Geel, Belgium)
STAIN (for cytogenetic assays): 5% (v/v) Giemsa (Merck) solution in Sörensenbuffer pH 6.8

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED: 1000 cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Evaluation criteria:

Biological relevance is evaluated against historical control ranges; any increase in aberrant cells remaining within the historical control data ranges is considered to be not biologically relevant.

Statistics:

A test substance is considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) 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, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics:

X2 = [(N-1) (ad-bc)2]/ (a+b) (c+d) (a+c) (b+d)]
where b = the total number of aberrant cells in the control cultures.
d = the total number of non aberrant cells in the control cultures.
n0 = the total number of cells scored in the control cultures.
a = the total number of aberrant cells in treated cultures to be compared with the control.
c = the total number of non aberrant cells in treated cultures to be compared with the control.
n1 = the total number of cells scored in the treated cultures.
N = sum of n0 and n1

If P [X2 > ((N-1) (ad-bc)2/(a+b) (c+d) (a+c) (b+d)] (one-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:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: The pH and osmolarity of a concentration of 5000 µg/ml were 8.04 and 313 mOsm/kg respectively (compared to 8.02 and 291 mOsm/kg in the solvent control).
- Water solubility: Solubility in vehicle Water: 20-23%, Culture medium: Not indicated
- Precipitation: A concentration of 5000 µg/ml showed no precipitation in the culture medium and was used as the highest concentration of K1600 black dye. It is possible that precipitate was not observed in the culture medium due to the dark colour of the test substance.

- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. K1600 black dye was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.

Lymphocytes (0.4 ml blood of a healthy 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 K1600 black dye for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A negative control was included at each exposure time.

The highest tested concentration was 5000 µg/ml.

After 3 h exposure to K1600 black dye in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 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).

Cytotoxicity of K1600 black dye in the lymphocyte cultures was determined using the mitotic index.

Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was the recommended 5000 µg/ml.


COMPARISON WITH HISTORICAL CONTROL DATA: 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 in the solvent control cultures was within the laboratory historical control data range

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Acceptability of the assay

A chromosome aberration test is considered acceptable if it meets the following criteria:

a) The number of chromosome aberrations found in the solvent control cultures should

reasonably be within the laboratory historical control data range (see APPENDIX 4).

b) The positive control substances should produce a statistically significant (Chi-square test,

one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

c) A homogeneous response between the replicate cultures is observed.

d) A possible precipitate present on the slides should not interfere with the scoring of chromosome

aberrations.

Data evaluation and statistical procedures

A test substance is considered positive (clastogenic) in the chromosome aberration test if:

a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in

the number of cells with chromosome aberrations.

b) 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, one-sided, p < 0.05)

increase in the number of cells with chromosome aberrations.

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or

excluded) for each exposure group outside the laboratory historical control data range was compared

to that of the solvent control using Chi-square statistics:

If P(one-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.

Biological relevance is evaluated against historical control ranges; any increase in aberrant cells

remaining within the historical control data ranges is considered to be not biologically relevant.

RESULTS

Dose range finding test

A concentration of 5000 µg/ml showed no precipitation in the culture medium and was used as the

highest concentration of K1600 black dye. It is possible that precipitate was not observed in the culture

medium due to the dark colour of the test substance.

The pH and osmolarity of a concentration of 5000 µg/ml were 8.04 and 313 mOsm/kg respectively

(compared to 8.02 and 291 mOsm/kg in the solvent control).

The dose range finding test blood cultures were treated with 52, 164, 512, 1600 and 5000 µg

K1600 black dye/ml culture medium with and without S9-mix.

Table 1 (APPENDIX 1) shows the mitotic index of cultures treated with various K1600 black dye

concentrations or with the negative control substance.

First cytogenetic assay

Based on the results of the dose range finding test the following dose levels were selected for the

cytogenetic assay:

Without and with S9-mix: 512, 1600 and 5000 µg/ml culture medium

(3 h exposure time, 24 h fixation time).

Table 2 (APPENDIX 1) shows the mitotic index of cultures treated with various K1600 black dye

concentrations or with the positive or negative control substances.

All dose levels were selected for scoring of chromosome aberrations. Both in the absence and

presence of S9-mix, K1600 black dye did not induce a statistically significant or biologically relevant

increase in the number of cells with chromosome aberrations (APPENDIX 1: Table 3, Table 4).

Both in the absence and presence of S9-mix, K1600 black dye did not increase the number of

polyploid cells and cells with endoreduplicated chromosomes.

Second cytogenetic assay

To obtain more information about the possible clastogenicity of K1600 black dye, a second cytogenetic

assay was performed in which human lymphocytes were continuously exposed to K1600 black dye in

the absence of S9-mix for 24 or 48 hours. The following dose levels were selected for the second

cytogenetic assay:

Without S9-mix : 500, 1600, 3000 and 5000 µg/ml culture medium

(24 h and 48 h exposure time, 24 h and 48 h fixation time).

Table 5 (APPENDIX 1) shows the mitotic index of cultures treated with various K1600 black dye

concentrations or with the positive or negative control substances.

Based on these observations the following doses were selected for scoring of chromosome

aberrations:

Without S9-mix : 500, 1600 and 5000 µg/ml culture medium

(24 h and 48 h exposure time, 24 h and 48 h fixation time).

K1600 black dye did not induce a statistically significant or biologically relevant increase in the number

of cells with chromosome aberrations (APPENDIX 1; Table 6 - 7).

K1600 black dye did not increase the number of polyploid cells and cells with endoreduplicated

chromosomes.

See attached document for tables

Applicant's summary and conclusion

Conclusions:

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

It is concluded that this test is valid and that K1600 black dye is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Executive summary:

Evaluation of the ability of K1600 black dye to induce chromosome aberrations in cultured peripheral human lymphocytes.

This report describes the effect of K1600 black dye on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity of K1600 black dye was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD, EC and MITI guidelines.

Batch G-152 of K1600 black dye was a black powder with a purity of 99.03%. K1600 black dye was suspended in culture medium.

In the first cytogenetic assay, K1600 black dye was tested up to 5000 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. This is the highest concentration recommended for testing in the guidelines.

In the second cytogenetic assay, K1600 black dye was also tested up to 5000 µg/ml for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

K1600 black dye did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

No effects of K1600 black dye 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 K1600 black dye does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

Finally, it is concluded that this test is valid and that K1600 black dye is not clastogenic in human lymphocytes under the experimental conditions described in this report.