Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 January 2017 - 28 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

26 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

dd 03 November 2015

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

EC Number:

227-497-9

EC Name:

Disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Cas Number:

5858-81-1

Molecular formula:

C18H12N2Na2O6S

IUPAC Name:

disodium 3-hydroxy-4-[(4-methyl-2-sulphonatophenyl)azo]-2-naphthoate

Test material form:

solid: particulate/powder

Details on test material:

Name as cited in the report: D&C Red 6
Appearance: red powder
Storage conditions: at room temperature

Specific details on test material used for the study:

No correction was made for purity.

Method

Vehicle / solvent:

- Vehicle used: RPMI 1640 medium
- Justification for choice of solvent/vehicle: a homogeneous suspension could be obtained by suspending the test substance in the vehicle

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in RPMI 1640 medium

A dose-range finding study was conducted as part of the first cytogenetic assay. To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix.

DURATION
- Preincubation period: 48 hr
- Exposure duration experiment 1: 3, 24 and 48 h (without S9) and 3 h (with S9)

- Exposure duration experiment 2: 24 and 48 h (without S9)
- Fixation time: 24 h (for 3 h exposure) and 24 and 48 h (for 24 and 48 h exposure)

SPINDLE INHIBITOR: colchicine (0.5 μg/mL medium)

STAIN: 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8

NUMBER OF REPLICATIONS: the test substance was tested in duplicate in two independent experiments

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: 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. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min. Slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene/pertex and mounted with a coverslip in an automated cover slipper.

NUMBER OF CELLS EVALUATED: 1000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 150 in each replicate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity: cytotoxicity of the test substance in the lymphocyte cultures was determined using the mitotic index. No cytotoxicity was observed in the duplicate cultures of the 3 h exposure time and the slides were scored for chromosome aberrations.

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

Evaluation criteria:

EVALUATION CRITERIA:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with an Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with an Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

ACCEPTABILITY CRITERIA:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analysed by the Fisher’s exact test (one-sided, p < 0.05).

Statistics:

Fisher's exact test, one-sided, was used to compare the incidence of abberant cells cells with the concurrent negative control. Cochran Armitage trend test was used to evaluate the dose-response relationship.
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) and ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) were used for statistical analysis of the data.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: cultured peripheral human 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:

- In the absence of S9-mix, D&C Red 6 induced a statistically significant increase in the number of cells with chromosome aberrations at the highest tested concentration only. However, since the mean number of aberrations was within the 95% control limits of the distribution of the historical negative control database and in addition a negative Cochran Armitage trend test was obtained (p = 0.146 and p=0.125 in the absence and presence of gaps respectively), this increase was considered not biologically significant.

- 1 endoreduplicated chromosome was observed at the lowest dose level at the 24 hours exposure time, which is outside the 95%
control limits of the distribution of the historical negative control database, this was considered an isolated event and therefore considered not biologically relevant.

RANGE-FINDING/SCREENING STUDIES: a dose-range finding study was conducted as part of the first cytogenetic experiment.

HISTORICAL CONTROL DATA
- Positive historical control data: the number of cells with chromosome aberrations found in the positive control cultures was within the
95% control limits of the distribution of the historical positive control database, as well as the mean number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures.
- Solvent historical control data: the number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database

Applicant's summary and conclusion

Conclusions:

A chromosome aberration study was performed with D&C Red 6, according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that D&C Red 6 is not clastogenic in human lymphocytes with and without metabolic activation.

Executive summary:

A chromosome aberration test was performed according to OECD guideline 473 and GLP principles, to assess the ability of D&C Red 6 to induce chromosome aberrations in cultured peripheral human lymphocytes. The possible clastogenicity of the test substance was tested with and without metabolic activation. The test was performed in duplicate in two independent experiments, including a positive control and a solvent control. Results from the positive and solvent control were within the 95% control limits of the distribution of the historical data range. The test substance did not induce biologically relevant increases in the number of cells with chromosome aberations, polyploid cells and cells with endoreduplicated chromosomes. Therefore it can be concluded that D&C Red 6 is not clastogenic in human lymphocytes.