Benzo[rst]phenanthro[10,1,2-cde]pentaphene-9,18-dione, reaction products with 1-chlorododecane

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:

November 3, 2016 to June 5, 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Chromosomal aberration assay utilizing rat lymphocytes.

Test material

Test material information

Constituent 1

Reference substance name:

Benzo[rst]phenanthro[10,1,2-cde]pentaphene-9,18-dione, reaction products with 1-chlorododecane

EC Number:

822-536-1

Cas Number:

2180952-76-3

Molecular formula:

na

IUPAC Name:

Benzo[rst]phenanthro[10,1,2-cde]pentaphene-9,18-dione, reaction products with 1-chlorododecane

Details on test material:

Lot #: ZA07262016

Specific details on test material used for the study:

Test Material Name: C.I. Solvent Red 175 Solid (solvent stripped)
Chemical Name: Dinaphtho(1,2,3-cd:1’,2’,3’-lm)perylene-9,18-dione, lauryl derivatives
Synonyms: None
Lot/Reference/Batch Number: ZA07262016
Purity/Characterization (Method of Analysis and Reference): The test material was determined to contain 93.9 ± 0.06 wt% active ingredient by difference (100 – wt% residual lauryl chloride) with identification by nuclear magnetic resonance spectroscopy and liquid chromatography mass spectrometry (Kiefer, 2017).
Test Material Stability Under Storage Conditions: C.I. Solvent Red 175 Solid (solvent stripped), lot ZA07262016, was determined to be stable for 2 weeks at 54°C which is equivalent to 24 months under ambient storage conditions as tested under USEPA OPPTS Guideline 830.6313 (Kiefer and Kerry, 2017).

Method

Target gene:

Chromosomal aberration assay utilizing rat lymphocytes.

Species / strain

Species / strain / cell type:

lymphocytes: Rat

Details on mammalian cell type (if applicable):

Species and Sex: Rats (Male)
Strain and Justification: Crl:CD(SD) Rats were selected because of their general acceptance and suitability for toxicity testing, availability of historical background data, and the reliability of the commercial supplier.
Supplier and Location: Charles River Laboratories (Kingston, New York)
Age at Study Start: 10 weeks

Lymphocyte Cultures:
The animals were euthanized with carbon dioxide just prior to collecting the samples by cardiac puncture. Blood, treated with an anticoagulant (e.g., heparin), and from several
rats was pooled for sample collection. 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 Lglutamine)
in addition with 40 μg/ml PHA-P. Cultures were initiated by inoculating
approximately 0.5 ml of whole blood into 5 ml of medium. Cultures were set up in
duplicate at each dose level in T-25 plastic tissue culture flasks and incubated at 37°C.
Treatment medium was the above-mentioned medium without serum and was used
during the 4-hour treatment conditions, while complete medium was used for the 24-hour
treatment condition.

Metabolic activation:

with and without

Metabolic activation system:

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

Test concentrations with justification for top dose:

Cells were treated either in the absence or presence of S9 activation with concentrations ranging from 0.0 (vehicle control) to 250.0 μg C.I. Solvent Red 175 Solid (solvent stripped) per ml of culture medium. The highest concentration was based on the limit of solubility of the test material in the treatment medium.

Vehicle / solvent:

Initially ethanol (CAS No. 64-17-5) was selected as the solvent used to dissolve the test material; however, upon further analysis, acetone (CAS No. 67-64-1) proved to be a better solvent to dissolve the test material and was used as the vehicle control.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: Cyclophosphamide monohydrate

Details on test system and experimental conditions:

Lymphocyte Cultures:
The animals were euthanized with carbon dioxide just prior to collecting the samples by cardiac puncture. Blood, treated with an anticoagulant (e.g., heparin), and from several rats was pooled for sample collection. Whole blood cultures were set up in complete medium (RPMI 1640 medium with 25 mM HEPES, supplemented with 10% heatinactivated 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 Lglutamine) in addition with 40 μg/ml PHA-P. Cultures were initiated by inoculating approximately 0.5 ml of whole blood into 5 ml of medium. Cultures were set up in duplicate at each dose level in T-25 plastic tissue culture flasks and incubated at 37°C. Treatment medium was the above-mentioned medium without serum and was used during the 4-hour treatment conditions, while complete medium was used for the 24-hour treatment condition.

In Vitro Metabolic Activation System:
S9 liver homogenate prepared from Aroclor 1254-treated (500 mg/kg body weight) male Sprague Dawley rats were purchased from a commercial source, and stored at -100°C or below. Thawed S9 was reconstituted at a final concentration of 10% (v/v) in a "mix" (O'Neill et al., 1982). The mix consisted of 10 mM MgCl2·6H2O, 5 mM glucose-6-phosphate, 4 mM nicotinamide adenine dinucleotide phosphate, 10 mM CaCl2, 30 mM KCl, and 50 mM sodium phosphate (pH 8.0). The reconstituted mix was added to the treatment medium to obtain the desired final concentration of S9 in the culture, i.e., 2% v/v. Hence, the final concentration of the co-factors in the medium was 1/5 of the concentrations stated above.

Preparation of the Treatment Solution and Administration of the Test Material:
The test material was found to be soluble in acetone up to 200.0 mg/ml. All test material solutions were prepared fresh on the day of treatment and used within two hours of preparation. The test material was dissolved in acetone and further diluted (1: 100) in medium. This technique has been shown to be an effective method for detecting various in vitro clastogens in this test system. All dosing units were expressed in μg/ml. MMC was dissolved in treatment medium, and CP stock was dissolved in distilled water.

Dose Level Selection:
The cultures were treated with various concentrations of the test material and the selected concentration of the positive control chemicals. Soluble materials were tested up to 10 mM, 2000 μg/ml, or 2 μl/ml, whichever was the lowest. Test materials with limited solubility were tested up to or beyond their limit of solubility. In some cases, more than one insoluble concentration was tested to ascertain whether toxicity would occur at higher insoluble concentrations. The other concentrations tested were separated by a factor of 2 to 3.

Analytical Verification of Dosing Solutions:
The selected concentrations of the test material in the stock dosing solutions used for treatment in Assay B1 were verified by the Analytical Chemistry Laboratory, Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan. Samples were diluted in an appropriate solvent and analyzed by high performance liquid chromatography with fluorescence detection (GC/FLD). Analytical method validation was performed concurrently with sample analysis. Homogeneity analysis was conducted, on all doses, as test material was administered as a suspension.

Identification of the Test System:
All test cultures were identified using self-adhesive labels containing a code system that identified the test material, experiment number, treatment, and replicate.

Evaluation criteria:

Evaluation Criteria:
For a test to be acceptable, the chromosomal aberration frequency in the positive control cultures should be significantly higher than the vehicle controls. The aberration frequency in the vehicle and positive controls should be within the control limits of the laboratory historical control values as calculated using previous laboratory values. A test chemical was considered positive in this assay if it induced a statistically significant, dose-related increase in the frequency of cells with aberrations and the incidence of aberrant cells was outside the control limits of the laboratory historical vehicle control range. A test chemical was considered negative in this assay if it did not induce a statistically significant, dose-related increase in the frequency of cells with aberrations and the incidence of aberrant cells was not outside the control limits of the laboratory historical vehicle control range. If a test chemical did not meet either of the above criteria it may have been considered equivocal.

Statistics:

Statistical Analysis:
The proportions of cells with aberrations (excluding gaps) were compared by the following statistical methods. At each dose level, data from the replicates were pooled. A two-way contingency table was constructed to analyze the frequencies of aberrant cells. An overall Chi-square statistic, based on the table, was partitioned into components of interest. Specifically, statistics were generated to test the global hypothesis of no difference in the average number of cells with aberrations among the dose groups (Armitage, 1971). An ordinal metric (0, 1, 2, etc.) was used for the doses in the statistical evaluation. If this statistic was found to be significant at alpha = 0.05, versus a one-sided increasing alternative, pairwise tests (i.e., control vs. treatment) were performed at each dose level and evaluated at alpha = 0.05, again versus a one-sided alternative. If any of the pairwise tests were significant, a test for linear trend of increasing number of cells with aberrations with increasing dose was performed (Armitage, 1971).
Polyploid cells were analyzed by the Fisher Exact probability test (Siegel, 1956). The number of polyploid cells were pooled across replicates for the analysis and evaluated at alpha = 0.05. The data was analyzed separately based on the presence or absence of S9 and based on the exposure time.

Results and discussion

Test results

Test results 1

Species / strain:

lymphocytes: Rat

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

No significant increases in the incidence of polyploid cells or frequency of cells with aberrations in the test material treated cultures (31.3, 62.5 and 125.0 μg/ml) as compared to the negative control and the vehicle control.

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: Rat

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

No significant increases in the incidence of polyploid cells or frequency of cells with aberrations in the test material treated cultures (31.3, 62.5 and 125.0 μg/ml) as compared to the negative control and the vehicle control.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Increasing toxicity was observed with increasing dose (See results discussion).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

lymphocytes: Rat

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

No significant increases in the incidence of polyploid cells or frequency of cells with aberrations in the test material treated cultures (31.3, 62.5 and 125.0 μg/ml) as compared to the negative control and the vehicle control.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Increasing toxicity was observed with increasing dose (See results discussion).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

pH and Osmolality:
The pH and osmolality of treatment medium containing approximately 2000.0 μg/ml of the test material and medium containing 1% (acetone) were determined using a Denver Basic pH meter (Denver Instrument Co., Arvada, Colorado) and an OSMETTE A freezing point osmometer (Precision Systems, Inc., Natick, Massachusetts), respectively. There was no appreciable change in either the pH or osmolality at this concentration as compared to the treatment medium with solvent alone (treatment medium with the test material, pH = 7.59, osmolality = 443 mOsm/kg H2O; treatment medium with 1% acetone, pH = 7.61, osmolality = 440 mOsm/kg H2O).

Remarks on result:

other: Assay B1 4-Hour Treatment

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), 3.3, 6.5, 13.0, 26.0, 52.1, 104.2, and 208.3 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 1.6 μg/ml. The test material precipitated in the treatment medium at the top concentration (i.e., 208.3 μg/ml) in all treatment conditions, as observed at the end of treatment. Due to poor lymphocyte growth in all cultures, the slides were not analyzable (data included in the study file) and all portions of this assay had to be repeated in a separate assay (Assay B1).

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), 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, and 250.0 μg/ml. Cultures were also treated continuously for 24 hours in the absence of S9 with the above concentrations plus an additional lower concentration (i.e., 2.0 μg/ml). The test material precipitated in the treatment medium at the top two concentrations (i.e., 125.0 and 250.0 μg/ml). Analytically detected concentrations of the test material in the stock solutions (Assay B1) varied from 99.8 to 138.9% of the target and verified that concentrations used for treatment were within the acceptable range.

Short Treatment:

In the absence of S9, the cultures displayed no toxicity with relative mitotic indices ranging from 93.6 to 118.3% compared to the vehicle control values. In the presence of S9, the mitotic indices of the treated cultures ranged from 66.2 to 100.0% as compared to the vehicle control values. Based upon these results, cultures treated with targeted concentrations of 0.0 (negative control), 0.0 (1% acetone, vehicle control), 31.3, 62.5, and 125.0 μg/ml were chosen for the determination of chromosomal aberration frequency and incidence of polyploidy both in the absence and presence of S9 activation.

Among the cultures treated with the positive control chemicals for 4 hours, 0.5 μg/ml of MMC and 2 μg/ml of CP were selected for evaluation of aberrations in the absence and presence of S9, respectively.

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 4-hour non-activation assay, the frequency of cells with aberrations in the negative control and vehicle control were 0.7% and 1.0%, respectively. The corresponding values at treatment levels of 31.3, 62.5, and 125.0 μg/ml were 0.7, 0.3, and 0.3%, respectively. In the activation assay, cultures treated with the test material at concentrations of 31.3, 62.5, and 125.0 μg/ml had aberrant cell frequencies of 0.3, 1.0 and 1.7%, respectively as compared to the negative control and vehicle control values of 0.7% and 0.7%, respectively. 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 chemicals. Aberrant cell frequencies in MMC (-S9, 4-hour treatment), and CP (+S9, 4-hour treatment) cultures were 28.0%, and 28.0%, respectively. 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 minimal to no toxicity, with relative mitotic indices ranging from 68.4 to 107.0% relative to the vehicle control value, although no dose response was observed. Based upon these results, cultures treated with targeted concentrations of 0.0 (negative control), 0.0 (1% acetone, vehicle control), 31.3, 62.5, and 125.0 μg/ml were chosen for the determination of chromosomal aberration frequencies and incidence of polyploidy. Cultures treated with 0.075 μg/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 negative control and vehicle control were 0.0% and 0.0%, respectively. The corresponding values at concentration levels of 31.3, 62.5, and 125.0 μg/ml were 0.0, 0.0, and 1.3%, respectively. 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 7.7%. 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 Red 175 Solid (solvent stripped) was negative in this in vitro chromosomal aberration test.

Executive summary:

C.I. Solvent Red 175 Solid (solvent stripped) (Dinaphtho(1,2,3-cd:1’,2’,3’-lm)perylene-9,18-dione, lauryl derivatives) was evaluated in an in 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 0.0 (vehicle control) to 250.0 μg C.I. Solvent Red 175 Solid (solvent stripped) 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 Red 175 Solid (solvent stripped) in the dose preparations ranged from 99.8 to 138.9% 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 the solubility of the test material. In this study, cultures treated for 4 hours with targeted concentrations of 0.0 (negative control), 0.0 (1% acetone, vehicle control), 31.3, 62.5, and 125.0 μg/ml in the absence and in the presence of S9 and cultures treated for 24 hours with 0 (vehicle control), 31.3, 62.5, and 125.0 μg/ml were analyzed.

There were no significant increases in the frequency of cells with aberrations administered C.I. Solvent Red 175 Solid (solvent stripped) 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

Red 175 Solid (solvent stripped) was considered to be negative in this in vitro chromosomal aberration assay utilizing rat lymphocytes.