Reaction mass of 1,4-bis(methylamino)anthraquinone and 1,4-bis[(2-ethylhexyl)amino]anthraquinone and 1-[(2-ethylhexyl)amino]-4-(methylamino)anthraquinone and 9,10-Anthracenedione, 1,4-bis(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-(methylamino)-4-(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-[(2-ethylhexyl)amino]-4-(pentylamino)-, branched and linear

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:

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In Vitro Chromosomal Aberration Assay Utilizing Rat Lymphocytes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 1,4-bis(methylamino)anthraquinone and 1,4-bis[(2-ethylhexyl)amino]anthraquinone and 1-[(2-ethylhexyl)amino]-4-(methylamino)anthraquinone and 9,10-Anthracenedione, 1,4-bis(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-(methylamino)-4-(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-[(2-ethylhexyl)amino]-4-(pentylamino)-, branched and linear

EC Number:

911-360-1

Molecular formula:

variable structures

IUPAC Name:

Reaction mass of 1,4-bis(methylamino)anthraquinone and 1,4-bis[(2-ethylhexyl)amino]anthraquinone and 1-[(2-ethylhexyl)amino]-4-(methylamino)anthraquinone and 9,10-Anthracenedione, 1,4-bis(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-(methylamino)-4-(pentylamino)-, branched and linear and 9,10-Anthracenedione, 1-[(2-ethylhexyl)amino]-4-(pentylamino)-, branched and linear

Method

Target gene:

NOt applicable

Species / strain

Species / strain / cell type:

lymphocytes: rat

Details on mammalian cell type (if applicable):

Strain of rats: Crl:CD(SD)
Sex: Male
Age at start of study: 10-16 weeks
Whole blood cultures were set up in complete medium (RPMI 1640 medium with 25 mM HEPES, supplemented with 10% heat-inactivated fetal bovine serum, antibiotics and antimycotics (penicillin G, 100 units/ml; streptomycin sulfate, 0.1 mg/ml; fungizone 0.25 µg/ml), and an additional 2 mM L-glutamine) in addition with 30 - 50 µg/ml PHA-P. Cultures were initiated by inoculating approximately 0.5 ml of whole blood into 5 ml of medium.

Cytokinesis block (if used):

Colcemid (1 microgram/culture)

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate prepared from Aroclor 1254-treated (500 mg/kg body weight) male Sprague Dawley

Test concentrations with justification for top dose:

Assay B1 (4hr treatment with and without metabolic activation, and 24 hours in the absence of metabolic activation:
Doses used were: 0 (vehicle control), 2.8, 5.6, 11.2, 22.3, 44.7, 89.3, and 178.6 µg/ml
Based on precipitation at the top two dose levels and the Mitotic indices scores, the following dose levels were selected for analysis of chromosomal aberration frequency for the 4hr treatment with S9 and the 24 hr treatment without S9: 0 (vehicle control), 2.8, 11.2, and 44.7 µg/ml
a technical error resulted in the 4hr treatment in the absence of s( being repeated in Assay C1.
Assay C1: 4hr treatment in the absence of s9 (repeat of this part of B1)
Doses used were: 0 (vehicle control), 2.8, 5.6, 11.2, 22.3, 44.7, 89.3, and 178.6 µg/ml
Dose levels of 0 (vehicle control), 22.3, 89.3, and 178.6 µg/ml were chosen for the determination of chromosomal aberration frequency and incidence of polyploidy in the absence of S9 activation.

Vehicle / solvent:

Ethanol (CAS No.64-17-5)

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol (CAS No.64-17-5)

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: rat

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

Positive controls validity:

valid

Any other information on results incl. tables

Assay A1

Cultures were treated with the test material for 4 hours in the absence and presence of S9 activation at concentrations of 0 (vehicle control), 5.6, 11.2, 22.3, 44.7, 89.3, 178.6, and 357.2 µg/ml. Cultures were also treated continuously for 24 hour in the absence of S9 with the above concentrations plus and additional lower concentration of 2.8mg/ml. The test material precipitated in the treatment medium at the top two concentrations (i.e., 178.6 and 357.2mg/ml in all treatment conditions as, observed at the end of treatment. Due to poor lymphocyte growth the slides in all conditions were not evaluated for mitotic indices or chromosome aberrations. As a result all conditions of this assay were repeated in a separate assay (Assay B1). Analytically detected concentrations of the test material in the stock solutions (Assay A1) varied from 105.0 to 142.0% of the target and verified that concentrations used for treatment were within the acceptable range. 

Assay B1

Cultures were treated with the test material for 4 hours in the absence and presence of S9 activation at concentrations of 0 (vehicle control), 2.8, 5.6, 11.2, 22.3, 44.7, 89.3, and 178.6 µg/ml. Cultures were also treated continuously for 24 hours in the absence of S9 with the above concentrations plus an additional lower concentration of 1.4mg/ml. The test material precipitated in the treatment medium at the top concentration (i.e., 178.6 µg/ml) in all treatment conditions, as observed at the end of treatment. 

Short Treatment

In the absence of S9, the cultures displayed no toxicity with relative mitotic indices ranging from 86.2 to 120.0% compared to the vehicle control values. However, due to a technical error in the absence of S9 the vehicle controls were unanalyzable for chromosomal aberrations and therefore, this portion of the assay was repeated in an independent assay. In the presence of S9, the mitotic indices of the treated cultures ranged from 17.1 to 75.0% as compared to the vehicle control values. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 2.8, 11.2, and 44.7 µg/ml were chosen for the determination of chromosomal aberration frequency and incidence of polyploidy in the presence of S9 activation. 

Among the cultures treated with the positive control chemicals for 4 hours, 2 µg/ml of CP were selected for evaluation of aberrations in the presence of S9.

There were no significant increases in the incidence of polyploid cells in any of the test material treated cultures as compared to the vehicle control values.

In the activation assay, cultures treated with the test material at concentrations of 2.8, 11.2, and 44.7 µg/ml had aberrant cell frequencies of 0.7, 2.7 and 3.0%, respectively as compared to the vehicle control value of 1.0%. Statistical analyses of these data did not identify significant differences between the vehicle control and any of the treated cultures without or with S9 activation. The frequencies of aberrant cells observed in the test material treated cultures were within the control limits of the laboratory historical vehicle control range.

Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemical. Aberrant cell frequencies in CP (+S9, 4-hour treatment) cultures were 31.3%. All values were within the control limits of the laboratory historical positive control range.

Continuous Treatment

Based upon the negative findings in the 4-hour treatment, slides from the continuous 24-hour treatment were evaluated. Cultures treated continuously for 24 hours in the absence of S9 activation had severe toxicity with relative mitotic indices ranging from 0.9 to 92.7% relative to the vehicle control value. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 2.8, 11.2, and 44.7mg/ml were chosen for the determination of chromosomal aberration frequencies and incidence of polyploidy. Cultures treated with 0.075mg/ml MMC were selected to serve as the positive control.

There were no significant increases in the incidence of polyploid cells in any of the test material treated cultures as compared to the vehicle control values.

The frequency of aberrant cells in the vehicle control was 0.3% and the corresponding values at concentration levels of 2.8, 11.2, and 44.7mg/ml were 0.0, 0.0, and 0.8%, respectively. As a result of the decreased relative mitotic index and limited number of mitotic figures present on the slides only 259 metaphase spreads were evaluated from the 44.7 µg/ml concentration and 272 metaphase spreads from the 0.075 µg/ml MMC concentration instead of the standard 300. This minor variation was deemed to be inconsequential to the interpretation of the study. There were no statistically significant differences between the test material treated cultures and the vehicle control values and all values were within the control limits of the laboratory historical vehicle control range.

A significant increase in the frequency of cells with aberrations was observed in cultures treated with the positive control chemical. Aberrant cell frequency in MMC treated cultures was 11.8%. All values were within the control limits of the laboratory historical positive control range.

Assay C1

Cultures were treated with the test material for 4 hours in the absence of S9 activation at concentrations of 0 (vehicle control), 2.8, 5.6, 11.2, 22.3, 44.7, 89.3, and 178.6 µg/ml. The test material precipitated in the treatment medium at the top concentration (i.e., 178.6 µg/ml), as observed at the end of treatment. 

Short Treatment

In the absence of S9, the cultures displayed no toxicity with relative mitotic indices ranging from 71.4 to 89.8% compared to the vehicle control values. Based upon these results, cultures treated with targeted concentrations of 0 (vehicle control), 22.3, 89.3, and 178.6 µg/ml were chosen for the determination of chromosomal aberration frequency and incidence of polyploidy in the absence of S9 activation.

Among the cultures treated with the positive control chemicals for 4 hours, 0.5 µg/ml of MMC was selected for evaluation of aberrations in the absence of S9.

There were no significant increases in the incidence of polyploid cells in any of the test material treated cultures as compared to the vehicle control values.

In the non-activation assay, cultures treated with the test material at concentrations of 22.3, 89.3, and 178.6 µg/ml had aberrant cell frequencies of 0.0, 0.0 and 0.0%, respectively as compared to the vehicle control value of 1.3%. Statistical analyses of these data did not identify significant differences between the vehicle control and any of the treated cultures without or with S9 activation. The frequencies of aberrant cells observed in the test material treated cultures were within the control limits of the laboratory historical vehicle control range.

Significant increases in the frequency of cells with aberrations were observed in cultures treated with the positive control chemical. Aberrant cell frequencies in MMC (-S9, 4-hour treatment) cultures were 27.3%. All values were within the control limits of the laboratory historical positive control range.

Applicant's summary and conclusion

Conclusions:

It was concluded that under the experimental conditions used, C.I. Solvent Blue 98 (3 Amine) was negative in this in vitro chromosomal aberration test.

Executive summary:

C.I. Solvent Blue 98 (3 Amine) (1,4-Diamino-9,10-anthracenedione N,N’-mixed 2-ethylhexyl and methyl and pentyl derivatives) was evaluated in anin vitro chromosomal aberration assay utilizing rat lymphocytes. Approximately 48 hours after the initiation of whole blood cultures, cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0 (vehicle control) to 178.6mg C.I. Solvent Blue 98 (3 Amine) per ml of culture medium. The highest concentration was based on the limit of solubility of the test material in the treatment medium. The analytically determined concentrations of C.I. Solvent Blue 98 (3 Amine) in the dose preparations ranged from 105.0 to 142.0% of the targeted values. The duration of treatment was 4 hours without and with S9 and 24 hours without S9.  Selection of concentrations for the determination of the incidence of chromosomal aberrations was based upon cytotoxicity and the solubility of the test material. In this study cultures treated for 4 hours with targeted concentrations of 0 (vehicle control), 22.3, 89.3, and 178.6 mg/ml in the absence of S9 and 0 (vehicle control), 2.8, 11.2, and 44.7 mg/ml in the presence of S9 and cultures treated for 24 hours with 0 (vehicle control), 2.8, 11.2, and 44.7mg/ml were analyzed.

There were no significant increases in the frequency of cells with aberrations administered C.I. Solvent Blue 98 (3 Amine) in either the absence or presence of S9 activation. Cultures treated with the positive control chemicals (i.e., mitomycin C without S9 and cyclophosphamide with S9) had significantly higher incidences of aberrant cells. Based upon these results, C.I. Solvent Blue 98 (3 Amine) was considered to be negative in this in vitro chromosomal aberration assayutilizing rat lymphocytes.