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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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.

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).

Target gene:

Chromosomal aberration assay utilizing rat lymphocytes.

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.

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.

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

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

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

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.

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

10-13-1983 to10-24-1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report. E. Coli was not tested in the experiment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine loci

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fraction

Test concentrations with justification for top dose:

50, 166.6, 500, 1666.6 and 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Hexanes

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Hexanes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine

Details on test system and experimental conditions:

Test Organism:
Salmonella typhimurium, strains TA1535, TA1537, TA1538, TA98 and TA100

Source of test organism:
Dr. Bruce N. Ames, University of California, Biochemistry Department, Berkeley, California 94720

Rationale for Test System:
Chemicals capable of inducing mutations have been shown to increase the reversion frequency at the histidine locus in selected tester strains of Salmonella typhimurium with and without the addition of a metabolic activation system.

Test organism storage and maintenance:
Frozen working stock cultures were prepared by scraping a wooden applicator stick over the surface of frozen Master cultures and inoculating the scrapings into 50 ml of Oxoid Broth #2 and grown for approximately 16 hours at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker. Following the 16 hour growth period, 1 ml aliquots of the culture was dispensed into Nunc vials marked with the particular strain and quick frozen in an ethanol-dry ice bath before being stored at a minimum of -60°C. In order to avoid the effect of surface thawing and re-freezing of frozen permanent vials of bacterial stock, frozen working stock cultures are employed as a source of inoculum for mutagenesis testing. Fresh cultures for mutagenesis testing were prepared by quick thawing a vial of frozen working stock cultures of each tester strain and transferring the culture to 50 m of Oxoid Nutrient Broth #2 and grown for approximately 16 hours (1-2 x 10^9 cells/ml) at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker . After the 16 hour incubation, samples of culture suspensions were diluted 1:4 in distilled water and optical densities were observed at 650 nm using a Beckman Model 35 Spectrophotometer. Historical data has shown that optical densities of 0.400 or greater are representative of cells in late exponential or early stationary phase of growth. Tester strains were checked concurrently for the presence of the appropriate genetic markers at the time of the assay.

Top Agar:
Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5 mM histidine and 0.5 mM biotin at a volume of 0.1 ml per ml of agar, and maintained at 45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher Isotemp R Dry Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. All compound-treated tubes and plates were done in triplicate. Using sterile technique, the following were added to each tube in the following order: 2 ml aliquots of top agar solution, 0.1 ml of tester strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden.

Metabolic Activation System:
Tubes requiring metabolic activation have, in addition to the preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from Aroclor 1254-treated Sprague Dawley rats. The activation system (S-9 mix) contained the following per ml:
0.4 M MgC12; 1.65 M KC1 20 ul
1 M Glucose - 6 - Phosphate 5 ul
0.1 M NADP 40 ul
0.2 M Phosphate buffer pH 7.4 500 ul
Sterile distilled H2O 355 ul
S-9 Fraction 80 ul

The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates were allowed to harden for several minutes. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting revertant colonies on an electronic colony counter interfaced with a computer.

Bacterial Contaminant Control:
To insure the quality of aseptic technique during the assay and also the sterility of solvents, compounds and equipment, standard contamination checks were conducted with the assay. These contamination checks included the top level of test substance, solvent, top agar and S-9 mix at the same volumes as in the assay. The test substance or S-9 mix were added to 2 ml of molten top agar supplemented with 0.5mM histidine - 0.5mM biotin and poured onto minimal glucose plates. Top agar alone was also plated on minimal glucose plates. All plating was done in triplicate. Plates were incubated for 48 hours at 37°C (± 2°C) and then scored for contamination.

Test Article Purity:
The identity, purity, quality, and strength of the test article is the responsibility of the sponsor.

Test Article Stability:
Precipitates were observed upon addition of the 1.6, 5, 16.6 and 50 mg/ml dose level solutions to the aqueous top agar. There was no apparent change in the physical state of the control articles during the assay.

Evaluation criteria:

Data Reporting:
In scoring the assay, the positive and negative controls were first evaluated. If the negative control values did not fall within the acceptable historical mean values, the remaining plates were not scored and the assay was repeated. A summary of the data are presented in the summary data sheet contained in this report.

Evaluation Criteria:
In most tests with the Salmonella/Microsome Assay, results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered positive. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Statistics:

A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. If the solvent control is within one standard deviation of the historical mean or control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered positive. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Screen:
The preliminary toxicity screen for the Ames Assay used two of the histidine auxotrophs of Salmonella typhimurium TA1538 and TA100. The preliminary toxicity screen was designed to determine at which levels the compound exhibits toxic effects to the Salmonella typhimurium tester strains. The test compound was prepared to a concentration of 50 mg/ml. Logarithmic dilutions of this stock solution were made in the appropriate solvent to give the following concentrations: 0.5, 1.6, 5 and 16.6 mg/ml. Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5mM histidine - 0.5mM biotin at a volume of 0.1 ml/ml of agar, and maintained at 45°C until used. Sterile glass tubes with kaputs were labeled and placed into a Fisher Isotemp R Dry Bath at 45°C. All control and treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube: 2 ml aliquots of top agar solution, 0.1 ml of tester strain and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria.
Test article Fluorescent Yellow 131, Lot #1577-123, exhibited no inhibition of bacterial lawn growth at the levels tested in the Preliminary Toxicity Screen.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation All strains at all dose levels.
negative without metabolic activation All strains at all dose levels.

There were no observed increases of mutation frequencies in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at doses of 50, 166.6, 500, 1666.6 and 5000 ug/plate.

Executive summary:

Test article, Fluorescent Yellow 131, Lot #1577-123, was received as a dark red liquid and was soluble in hexane. Dose levels in a Preliminary Toxicity Screen were 50, 166.6, 500, 1666.6 and 5000 ug/plate. Strains TA1538 and TA100 of Salmonella typhimurium showed no inhibition of bacterial growth at the levels tested.

Test article Fluorescent Yellow 131 was evaluated in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at doses of 50, 166.6, 500, 1666.6 and 5000 ug/plate. There were 0.08 ml of S-9 supernatant (32.3 mg protein per ml) per 1.0 ml of S-9 mix in the rat liver metabolic activation preparation.

The results for test article Fluorescent Yellow 131, Lot #1577-123, were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at doses of 50, 166.6, 500, 1666.6 and 5000 ug/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable range of mean historical data.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6-14-2014 to 8-7-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

OECD guideline study performed in accordance with GLP. There were two deviations noted. One deviation from the protocol noted that the draft report date originally given was "latest by July, 2014. The actual draft report date was August 04, 2014. The second deviation noted was a deviation from GLP whereby it stated that Tetrahydrofuran would be used as the vehicle instead of DMSO, which was previously used in the study. However, 20% tetrahydrofuran in dimethyl sulfoxide was used as the vehicle in the study, based on the toxicity data of tetrahydrofuran generated before conducting initial toxicity test. This planned change was not documented before performing the activity. This error was not noticed until completion of the study. It was declared that these deviations did not affect the outcome of the study or interpretations of the results.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: METI:Reverse Mutagenicity Test on Bacteria, Methods of Testing New Chemical Substances (Section 5.1-1 to 5.1-11): Japanese Act on the Evaluation of Chemical Substances and Regulation of Their Manufacture, Act No. 117.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

This assay measures the ability of the test item to induce reverse mutations at specific histidine loci in the tester strains of Salmonella typhimurium i.e., TA1537, TA1535, TA98, TA100 and at tryptophan locus in Escherichia coli WP2 uvrA (pKM101).

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fraction

Test concentrations with justification for top dose:

The tester strains were exposed at the concentrations of 156.25, 312.5, 625, 1250, 2500 and 5000 μg C.I. Solvent Red 175/plate both in the absence and presence (5% v/v S9 mix) of metabolic activation for confirmatory mutation assay.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 20% tetrahydrofuran in dimethyl sulfoxide (20% THF + 80% DMSO) was selected as the vehicle for this study. This is also referred to as the "Negative Control" in this study.
- Justification for choice of solvent/vehicle: Solubility and precipitation tests of the test item were performed prior to the cytotoxicity test. The test item was insoluble in sterile distilled water and partially soluble in DMSO. An initial toxicity test and confirmatory mutagenicity test were conducted using DMSO as the solvent. Due to analytical recovery problems using DMSO as the solvent, the test item was tested for its solubility in tetrahydrofuran (THF), as recommended by the sponsor, and found to be soluble in THF. Due to expected toxicity with use of 100% THF as a solvent, solubility was also tested between 1-20% THF (prepared in DMSO). The test item formed a solution at 10% and 20% THF (prepared in DMSO). At the tested concentrations between (156.25 to 5000 μg/plate), the test item
formed an emulsion after addition into the top agar tubes (separation from aqueous phase) and precipitation (uneven distribution on the agar plates) was observed on the Minimal Glucose Agar (MGA) plates. These observations were noted after preparation of test item in THF only, as well as in 20% THF + 80% DMSO. Precipitation (uneven distribution of test item due to separation of test item from top agar) was observed at the concentration of 5000 μg/plate. Plates were checked visually and it was found that precipitation did not interfere with the counting of colonies. Therefore, 5000 μg/plate was selected as the highest concentration to be tested for initial toxicity test both in the absence and presence (5% v/v S9 mix) of metabolic activation. In pre-study screening, cytotoxicity was obsered in the THF treated plates at 80% and 100% THF, while cytotoxicity was not observed in 20% THF + 80% DMSO. So to avoid toxicity due to tetrahydrofuran (100% THF), 20% tetrahydrofuran in dimethyl sulfoxide (20% THF + 80% DMSO) was selected as the vehicle for this study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

20% THF + 80% DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: 2-aminoanthracene, 9-Aminoacridine hydrochloride hydrate

Details on test system and experimental conditions:

METHOD OF APPLICATION: Preincubation Assay Method

TEST SYSTEM:
The approved test system under the 1997 guidance of OECD allows the use of the following strains of bacteria:
Histidine auxotrophic strains of Salmonella typhimurium viz., TA98, TA100, TA1535 and TA1537.
Tryptophan auxotrophic strain of Escherichia coli WP2uvrA (pKM101).

Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision-repair system. Tester strains TA98 and TA100 also contain the pKM101 plasmid (carrying the R-factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.
TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshift and base substitution mutagens and TA1535 is reverted only by mutagens that cause base substitutions.
The E. coli tester strain has an AT base pair at the critical mutation site within the trpE gene (Wilcox et al., 1990). Tester strain WP2uvrA (pKM101) has a deletion in the uvrA gene resulting in a deficient DNA excision-repair system. Tryptophan revertants can arise due to a base change at the originally mutated site or by a base change elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base-pair substitution mutations (Green and Muriel, 1976).

Source:
The Salmonella typhimurium strains used in this study were mutants derived from Salmonella typhimurium LT2. The strains (Salmonella typhimurium TA1537, TA1535, TA98, TA100 and Escherichia coli WP2 uvrA (pKM101)) used in the study were obtained from Molecular Toxicology, Inc., 157 Industrial Park Dr, Boone, NC 28607 U.S.A.

Metabolic Activation System (S9 Fraction and S9 Mix):
A metabolically active extract of rat liver (treated with Aroclor 1254) called S9 fraction was used. The S9 fraction was buffered and supplemented with the essential co-factors β-NADP and D-Glucose-6-phosphate to form the “S9 mix”. This mix was added to the top agar in this assay. Aroclor 1254 induced rat liver S9 fraction (Maron and Ames, 1983; Venitt, Crofton-Sleigh, and Forster, 1984) procured from Molecular Toxicology Inc., U.S.A. (Lot No 3246) (APPENDIX 9) was used in the study.

TEST MEDIUM, SOLUTIONS AND OTHER CHEMICALS:
1 VB (Minimal Glucose Agar) agar
2 Soft agar
3 Soft agar (Top agar) containing 0.5 mM histidine and biotin
4 Soft agar containing 5 μg/mL tryptophan
5 Nutrient agar
6 Oxoid Nutrient broth No. 2
7 0.2 M sodium phosphate buffer

Incubation Time: 48 hours of incubation at 37 ± 1 °C

Number of replicates: Triplicate set of plates were treated for each test concentration of C.I. Solvent Red 175, negative and positive controls.

Evaluation criteria:

Cytotoxicity:
Five non-toxic doses were available to evaluate assay data. Cytotoxicity is detectable as more than 50% reduction in mean revertants per concentration relative to the mean negative control value (Teo et al., 2003) and by thinning of the bacterial background lawn.

Genotoxicity:
Assay Evaluation Criteria:
Once criteria for a valid assay have been met, responses observed in the assay were evaluated. The conditions necessary for determining a positive result were that there should be a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing doses of the test article either in the absence or presence of the metabolic activation system.

Strains TA98, TA1535, and TA1537:
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean negative control value.

Strains TA100 and Escherichia coli WP2 uvrA (pKM101):
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0- times the mean negative control value.

A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was determined to be non mutagenic.

Statistics:

Strains TA98, TA1535, and TA1537:
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean negative control value.

Strains TA100 and Escherichia coli WP2 uvrA (pKM101):
Data sets were judged positive, if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0- times the mean negative control value.

A response that did not meet all three of the above criteria (magnitude, concentration-responsiveness, reproducibility) was determined to be non mutagenic.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

All dose levels

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

All dose levels

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

5000 μg/plate

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

5000 μg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

5000 μg/plate

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

All dose levels

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

All dose levels

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

All dose levels

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: other: Initial Toxicity test

Remarks:

Migrated from field 'Test system'.

Negative Control:

The results of the study indicate that the values for the negative control (solvent/vehicle) (20% THF + 80% DMSO) in all strains were within limits of historical ranges for DMSO as well as distilled water. Results of the concurrent negative control (20% THF + 80% DMSO) were comparable with the results of concurrently run additional negative control (distilled water).

Positive Controls:

2-Aminoanthracene was used as the positive control in the presence of a metabolic activation for all the tester strains during the initial toxicity test and confirmatory mutation test. Historical control data of this laboratory proved the efficiency and suitability of 2-aminoanthracene as a positive control in the presence of metabolic activation. The batch of S9 used in this study was characterised with benzo(a)pyrene that requires metabolic activation by microsomal enzymes. Benzo(a)pyrene exhibited a clear increase in the number of revertants when compared with the concurrent negative control which demonstrated the efficiency of S9 used in this study.

Positive controls (both in the absence and presence of metabolic activation during both the trials) exhibited a clear increase in the number of revertants when compared with the concurrent negative control and were within the historical ranges. This demonstrated the efficiency of the test system and suitability of the procedures employed in the assay.

An increase in the mean number of revertants was not observed in Salmonella typhimurium tester strain TA100 (initial toxicity test and confirmatory mutation test) treated with 2-aminoanthracene in the absence of metabolic activation, but a clear increase was observed in the presence of metabolic activation. This demonstrated the efficiency of the S9 fraction used in this assay.

Initial Toxicity Test:

Partial inhibition of background lawn with reduction in revertant colonies was observed at the tested concentration of 5000 μg/plate both in the absence and presence of metabolic activation in all the Salmonella typhimurium tester strains and Escherichia coli WP2 uvrA (pKM101). Percent reduction observed was 45% and 36% in TA1537, 37% and 41% in TA1535, 47% and 39% in TA98, 34% and 40% in TA100 and 47% and 43% in E.coli WP2 uvrA (pKM101) in the absence and presence of metabolic activation system, respectively. Normal growth was observed up to the tested concentration of 1500 μg/plate both in the absence and presence of metabolic activation system.

Hence, 5000 μg C.I. Solvent Red 175/plate was selected as the highest concentration to be tested in the confirmatory mutagenicity assay both in the absence and presence of metabolic activation system, respectively, for Salmonella typhimurium tester strains and Escherichia coli WP2 uvrA (pKM101) tester strains.

Dose Formulation Analysis:

C. I. Solvent Red 175 was found to be within acceptable range of ± 15% of nominal (% RSD < 10%) in 20% THF + 80% DMSO at all the tested concentrations during confirmatory mutation assay. The doses complied with the presence of test item for its claimed concentration (± 15% of nominal) of active ingredient. Average recovery was 97.45%, 95.83%, 98.13%, 99.19%, 95.49% and 105.02% at test concentrations of 1562.5, 3125, 6250, 12500, 25000 and 50000 μg/mL, respectively. Average recovery for stability samples at the end of exposure was 97.13% and 105.08% at test concentrations of 1562.5 and 50000 μg/mL, respectively.

Confirmatory Mutation Assay:

Partial inhibition of bacterial lawn with reduction in number of revertant colonies was observed at the tested concentration of 5000 μg/plate in Salmonella typhimurium strains TA1537, TA1535 TA98, TA100 and Escherichia coli WP2 uvrA (pKM101) both in the absence and presence of metabolic activation (5% v/v S9 mix) when compared with the concurrent negative control. Percent reduction observed was 33% and 39% in TA1537, 64% and 42% in TA1535, 43% and 44% in TA98, 37% and 38% in TA100 and 38% and 27% in E. coli WP2 uvrA (pKM101) in the absence and presence of metabolic activation system, respectively.

Normal growth was observed up to the dose of 2500 μg/plate both in the absence and presence of the metabolic activation system (5% v/v S9 mix) in all the Salmonella typhimurium tester strains and Escherichia coli WP2 uvrA (pKM101).

No positive increase in the number of revertant colonies was observed in any of the tester strains at any of the tested concentrations when compared with the concurrent negative control. Results revealed that there was no positive mutagenic effect in Salmonella typhimurium strains TA1537, TA1535, TA98, TA100 and Escherichia coli WP2 uvrA (pKM101) at the test concentrations of

156.25, 312.5, 625, 1250, 2500 and 5000 μg C. I. Solvent Red 175/plate both in the absence and presence (5% v/v S9 mix) of metabolic activation when compared with the concurrent negative controls.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation All strains at all dose levels.
negative without metabolic activation All strains at all dose levels.

From the results of this study, under the specified experimental conditions, C.I. Solvent Red 175 was concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2 uvrA (pKM101).

Executive summary:

The potential of the C.I. Solvent Red 175 to induce reverse mutations in Salmonella typhimurium (strains: TA1537, TA1535, TA98 and TA100) and a tryptophan deficient strain, Escherichia coli WP2 uvrA (pKM101) was evaluated in the bacterial reverse mutation test. C.I. Solvent Red 175 was tested with and without metabolic activation using 20% tetrahydrofuran (THF) + 80% dimethyl sulfoxide (DMSO) as the solvent. Bacterial cultures were exposed to C.I. Solvent Red 175 at 6 concentrations (three plates/concentration) between 156.25 and 5000 μg/plate. From the results of the initial toxicity test, 5000 μg/plate was selected, being the recommended top dose indicated in the guidelines for testing and showing moderate cytotoxicity. After 48 hours of incubation at 37 ± 1 °C, the revertant colonies were scored.

C.I. Solvent Red 175 did not induce any significant increase in the number of revertants, in both the initial toxicity test and confirmatory mutation test, with and without S9 mix, in any of the five strains. All the values for the negative control were within historical control ranges of the laboratory and positive controls showed an increase in the number of revertant colonies, demonstrating the efficiency of the test system.

All criteria for a valid study were met as described in the protocol. From the results of this study, under the specified experimental conditions, C.I. Solvent Red 175 is concluded to be non-mutagenic in the Bacterial Reverse Mutation Assay using Salmonella typhimurium and Escherichia coli WP2 uvrA (pKM101). C.I. Solvent Red 175 was found to be within acceptable range of ± 15% of nominal concentrations at the tested stock concentrations of 1562.5, 3125, 6250, 12500, 25000 and 50000 μg/mL during the confirmatory mutation test. Average recovery was between 95% to 105% at the above tested concentration. Results of stability analysis showed that the test item was stable at the end of dosing (4 hours) and recovery of the test concentrations 1562.5 and 50000 μg/mL (highest and lowest concentration) was between 97-105%. Therefore, the doses complied with the presence of test item for claimed concentration (± 15 %) of active ingredient.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: (CHO/HGPRT) forward gene mutation assay

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
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 U.S. EPA OPPTS Guideline 830.6313 (Kiefer and Kerry, 2017).

Target gene:

The gene for Hgprt is located on the mammalian X-chromosome.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Indicator Cells and Justification of Their Use:
The cell line CHO-K1-BH4, originally obtained from Dr. Abraham Hsie, Oak Ridge National Laboratory, Oak Ridge, Tennessee, was used in this study. The CHO-K1-BH4 cell line was selected as the test system for this study because it is sensitive to mutagens, has a low background mutant frequency, and is readily available. Stock cultures were stored at approximately -80°C or below. The cultures were periodically checked for mycoplasma contamination (American Type Culture Collection, Manassas, Virginia). The cells were grown as monolayer cultures in plastic disposable tissue culture lab-ware under standard conditions of approximately 5% CO2 in air at 37°C in a humidified incubator.

Media:
The cells were routinely maintained in Ham's F-12 nutrient mix supplemented with 5% (v/v) heat-inactivated (56°C, 30 minutes), dialyzed fetal bovine serum, 25 mM HEPES, 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. Treatment medium was the above-mentioned medium without serum. The selection medium used for the detection of Hgprt- mutants was Ham's F-12 nutrient mix without hypoxanthine, supplemented with 10 µM 6 thioguanine, 5% serum, 25 mM HEPES, 2 mM L-glutamine, and the above-mentioned antibiotics.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats.

Test concentrations with justification for top dose:

The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation system (S9). The concentrations ranged from 15.6 to 250 µg/ml in the absence and presence of S9. The highest concentration was based on the limit of solubility of the test material in the treatment medium.

Vehicle / solvent:

Acetone (CAS No. 67-64-1) was selected as the solvent used to dissolve the test material and was used as the vehicle control.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: 20-methylcholanthrene

Details on test system and experimental conditions:

Controls:
Acetone (CAS No. 67-64-1) was selected as the solvent used to dissolve the test material and was used as the vehicle control. An untreated vehicle control was also utilized to verify the adequacy of the solvent within the test system. Ethyl methanesulfonate (EMS, CAS No. 62-50-0) was used as the positive control for the non-activation system (without S9 factor) at a final concentration of 621 µg/ml. The positive control for assays performed with S9 (activation system) was 20-methylcholanthrene (20-MCA, CAS No. 56-49-5) at concentrations of 4 and 8 µg/ml. The dose levels of EMS and 20-MCA were based upon our unpublished findings.

Preparation of the Treatment Solution and Administration of the Test Material:
The test material was found to be a workable suspension in acetone up to 200.0 mg/ml. All test material solutions were prepared fresh on the day of treatment and used within one hour of preparation. The test material was first dissolved in acetone and further diluted (1: 100 or 1:10) in medium to obtain the desired concentrations as recommended in the test guidelines. This technique has been shown to be an effective method for detecting various chemical mutagens in this test system (Hsie et al., 1981). EMS was dissolved in treatment medium. 20-MCA was dissolved first in DMSO and further diluted in the treatment medium. All dosing units were expressed in µg/ml.

Treatment Procedure:
Cells in logarithmic growth phase were trypsinized and placed in medium containing 5% serum at a standard density of 3.0 x 10^6 cells/T-75 flask approximately 24 hours prior to treatment. At the time of treatment, the culture medium was replaced with treatment medium, S9 mix (when applicable), the test chemical, the vehicle control, or the positive control chemical. The cells were treated for approximately 4 hours at 37°C and the exposure was terminated by washing the cells with phosphate buffered saline (Ca++ and Mg++ free).

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.

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 (HPLC/FLD). Analytical method validation was performed concurrently with sample analysis. Homogeneity analysis was conducted on the stock concentrations as the test material was administered as a suspension.

In Vitro Metabolic Activation:
S9 liver homogenates prepared from Aroclor 1254-induced male Sprague-Dawley rats were purchased from a commercial source and stored at approximately -80°C or below. Thawed S9 was reconstituted at a final concentration of 10% (v/v) in a “mix” (O’Neill et al., 1982). The S9 mix consisted of the following co-factors: 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 concentra¬tion of S9 in the culture, i.e., 2% v/v. Hence, the final concentration of the co-factors in the medium is 1/5 of the concentrations stated above.

Evaluation criteria:

Evaluation Criteria:
For an assay to be acceptable, the mutant frequency in positive controls should be significantly higher than the vehicle controls. The mutation 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. The test chemical was considered positive if it induced a statistically significant, dose-related increase in mutant frequency, and the mutant frequency was outside the control limit of the laboratory historical vehicle control range. The test chemical was considered negative if it did not induce a statistically significant, dose-related increase in mutant frequency, and the mutant frequency 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. The final interpretation of the data took into consideration such factors as the mutant frequency and cloning efficiencies in the vehicle and positive controls.

Statistics:

Statistical Analysis:
The frequency of mutants per 10^6 clonable cells was statistically evaluated using a weighted analysis of variance; weights were derived from the inverse of the mutant frequency variance. The actual plate counts were assumed to follow a Poisson distribution; therefore, the mean plate count was used as an estimate of variance (Kirkland, 1989).
If the analysis of variance was significant at alpha = 0.05, a Dunnett's t-test was conducted (Winer, 1971), comparing each treated group and the positive control to the solvent control (alpha = 0.05, one-sided). Linear dose-related trend tests were performed if any of the pairwise comparisons of test material with the solvent control yielded significant differences.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

The mutant frequencies observed in cultures treated with the test material in the absence and presence of S9 at the remaining concentration levels were not significantly different from the concurrent vehicle control values.

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

In the initial mutagenicity assay (Assay B1), in the absence of S9, no toxicity was observed with RS values ranging from 105.3 to 157.9%. In the presence of S9, no toxicity was observed with RS values ranging from 69.5 to 120.8%.

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

The mutant frequencies observed in cultures treated with the test material in the absence of S9 and presence of S9 were not significantly different from the concurrent vehicle control values.

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

There was no toxicity observed, as indicated by RS values, in the absence of S9 activation (84.0 to 101.5%). In the presence of S9, no toxicity was observed with RS values ranging from 69.6 to 97.3%.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

pH and Osmolality:
The pH and osmolality of treatment medium containing approximately 500.0 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). Alterations in the pH and osmolality of the culture medium have been shown to induce false positive responses in in vitro genotoxicity assays (Thilagar et al., 1984; Galloway et al., 1985; Cifone, 1985). 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 = 7.60, osmolality = 431 mOsm/kgH2O; treatment medium with 1% acetone, pH = 7.63, osmolality = 427 mOsm/kgH2O).

Remarks on result:

other: Assay B1 - Initial Mutagenicity Assay

Assay A1 - Preliminary Toxicity Assay:

In the preliminary toxicity assay, the test material was evaluated at concentrations of 0 (untreated vehicle control), 0 (vehicle control), 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125.0, and 250.0 µg/ml in the absence and presence of an externally supplied metabolic activation system (S9). The highest concentration tested was based upon the limitations imposed by the solubility of the test material in treatment medium. The highest two concentrations (125.0 and 250.0 µg/ml) precipitated in the treatment medium, as observed at the end of treatment. Although two precipitating concentrations were analyzed, the physical appearance differs between 125.0 and 250.0 µg/ml as the 125 µg/ml precipitating dose was barely visible. The treated cultures without S9 activation showed no toxicity with the relative survival (RS) values ranging from 78.7 to 98.2%. In the presence of S9 activation no toxicity was observed with RS values ranging from 74.7 to 102.7%. Based upon the results of this assay, concentration levels of 0 (untreated vehicle control), 0 (vehicle control), 15.6, 31.3, 62.5, 125.0, and 250.0 µg/ml of the test material were selected for the initial gene mutation assay in the absence and presence of S9.

Assay B1 - Initial Mutagenicity Assay:

In the initial mutagenicity assay (Assay B1), in the absence of S9, no toxicity was observed with RS values ranging from 105.3 to 157.9%. In the presence of S9, no toxicity was observed with RS values ranging from 69.5 to 120.8%. The mutant frequencies observed in cultures treated with the test material in the absence and presence of S9 at the remaining concentration levels were not significantly different from the concurrent vehicle control values. All mutant frequencies were within the control limits of the laboratory historical vehicle control range.

Assay C1 - Confirmatory Mutagenicity Assay:

In the confirmatory assay (Assay C1), the concentrations ranged from 15.6 to 250.0 µg/ml in the absence and presence of S9. There was no toxicity observed, as indicated by RS values, in the absence of S9 activation (84.0 to 101.5%). In the presence of S9, no toxicity was observed with RS values ranging from 69.6 to 97.3%. The mutant frequencies observed in cultures treated with the test material in the absence of S9 and presence of S9 were not significantly different from the concurrent vehicle control values. All average mutant frequencies were within the control limits of the laboratory historical vehicle control range.

In both the initial and confirmatory mutagenicity assays, the positive control chemicals induced significant increases in mutation frequencies and this data confirmed the adequacy of the experimental conditions for detecting induced mutations. The mutant frequencies exhibited by the positive control chemicals were either within or exceeded the control limits of the laboratory historical positive control range, with the exception of one of the two replicates in the 8 µg/ml concentration of MCA in assay B1 (presence of S9). As a result of this one replicate value the average mutant frequency of the 8 µg/ml MCA was below the control limits of the laboratory historical positive control range; however, the average mutant frequency was statistically increased when compared to the vehicle control. Furthermore, the 4 µg/ml MCA concentration of this assay was within the control limits of the laboratory historical positive control range verifying the validity of this assay.

The analytically observed concentrations of the test material in the stock dosing solutions in Assay B1 ranged from 90.5 to 108.1% of target and verified that concentrations used for treatment were within acceptable range.

Conclusions:

It was concluded that under the experimental conditions used, C.I. Solvent Red 175 Solid (solvent stripped) was negative in this in vitro CHO/HGPRT forward gene mutation assay.

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 the in vitro Chinese Hamster Ovary cell/hypoxanthine-guanine-phosphoribosyl transferase (CHO/HGPRT) forward gene mutation assay. The genotoxic potential of the test material was assessed in two independent assays in the absence and presence of an externally supplied metabolic activation system (S9). The concentrations ranged from 15.6 to 250 µg/ml in the absence and presence of S9. 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 90.5 to 108.1%. The adequacy of the experimental conditions for detection of induced mutation was confirmed by employing positive control chemicals, ethyl methanesulfonate for assays in the absence of S9 and 20-methylcholanthrene for assays in the presence of S9. Vehicle control cultures were treated with the solvent used to dissolve the test material (i.e.,acetone) and an untreated vehicle control was utilized to verify the adequacy of the solvent.

There were no statistically significant treatment-related increases in the mutant frequency in the test material-treated cultures compared to the vehicle control cultures in either the absence or presence of S9. Cultures treated with the positive control chemicals had significantly higher mutant frequencies. Based upon these results, C.I. Solvent Red 175 Solid (solvent stripped) was considered to be negative in this in vitro CHO/HGPRT forward gene mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 27, 1992 to September 14, 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

other: Bone marrow erythrocyte micronucleus assay

Specific details on test material used for the study:

Name: Fluorescent Yellow 131 SC (non-volatiles).
Lot No.: 7932-157.
Purity: Responsibility of the Sponsor.
Appearance and physical state at room temperature: Dark red liquid.
Stability: Responsibility of the Sponsor.
Storage conditions: Stored at room temperature in a plastic secondary container at the SRI Chemical Repository, Building M, Room 217.
Received: April 30, 1992.
Amount received at SRI: 100 grams.
Supplier: Morton International.
Expiration: Responsibility of the Sponsor.
Disposition: Remaining unused portion of the test article will be returned to the Sponsor unless otherwise stated. Dosing solutions were disposed at SRI.

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMAL SPECIFICATION:
Dose Range Finding Assay:
Swiss-Webster mice, 105 males and 105 females, born on approximately April 10, 1992 and March 14, 1992, respectively, were received by the SRI Laboratory Animal Medicine Department (LAMD) from Charles Rivers Laboratories (P01) on May 19, 1992. Upon arrival, mice of the same sex were randomly placed in animal cages (10 per cage) and 21 male and 21 female mice were selected randomly to be weighed. The weights of the male mice ranged from 17.6 to 26.1 g, and those of the female mice ranged from 16.4 to 24.7 g at the time of receipt. The weighed mice constitute 20% of the total number of mice of each sex received.
Eighteen male and 18 female mice were used for the dose range finding assay initiated on May 26, 1992. Mice not used for this particular study were assigned to concurrent studies.

Definitive Assay:
Swiss-Webster mice, 234 males and 195 females, born on approximately April 7, 1992 and March 5, 1992, respectively, were received by the SRI Laboratory Animal Medicine Department (LAMD) from Charles Rivers Laboratories (P01) on June 2, 1992. Upon arrival, mice of the same sex were randomly placed in animal cages (10 per cage) and 46 male and 40 female mice were selected randomly to be weighed. The weights of the male mice ranged from 20.1 to 27.5 g, and those of the female mice ranged from 21.2 to 28.3 g at the time of receipt. The weighed mice constitute 20% of the total number of mice of each sex received.
Fifty male and 50 female mice were used for the definitive assay initiated on June 16, 1992. Mice not used for this particular study were assigned to concurrent studies.

TEST SYSTEM IDENTIFICATION:
The animals were randomized and uniquely identified by ear punch. A card attached to the outside of each cage listed the project number, test article, dose level, time of sacrifice, sex, animal number, ear punch number, and tail marking.

SUPPLIER:
Charles River Laboratories
(Portage P01)
Shaver Road
Portage, Michigan 49081.

QUARANTINE:
Mice received on May 19, 1992 and June 2, 1992 were quarantined for 7 and 6 days, respectively, and released to the study on May 26, 1992 and June 8, 1992 by LAMD. Composite parasitology exams performed on May 26, 1992 and June 3, 1992 were negative, and no evidence of adverse clinical signs was observed during the two quarantine periods. No notable gross pathologies were found in any of the 8 male and 8 female mice sacrificed and necropsied during the quarantine periods for preliminary health evaluation.

ANIMAL ROOM ENVIRONMENTAL CONDITIONS:
Rooms: Building L, Room B134 for quarantines and Room W107 at SRI for dose range finding and definitive assays.
Temperature range: 68°-75°F.
Humidity range: 44-87%.
Light cycle: 12 hours light/12 hours dark.
Cage specification: Mice were housed no more than 10 to a cage during quarantine, 3 to a cage during the dose range finding assay, and 5 to a cage during the definitive assay. Polycarbonate cages with hardwood-chip bedding were used throughout the study.
There were no alterations in environmental conditions that could be expected to alter the results of this study.

FOOD AND WATER SUPPLY:
Food: Purina Certified Rodent Chow #5002 ad libitum. Purina Mills, Inc., St. Louis, MO. Lot Nos. FEB12921B and MAR11921C.
Water: UV purified/deionized tap water ad libitum was provided via an automatic watering system for quarantine and the definitive assay and via water bottles for the dose range finding assay. Water purity is analyzed quarterly, and results are recorded in Building 205, Room 39 at SRI.
There were no known contaminants in the water or diet that could be expected to alter the results of this study.

Route of administration:

oral: gavage

Vehicle:

VEHICLE CONTROL/DILUENT:
Name: Corn oil.
Lot No.: OCT0893A.
Purity: 100%, as per bottle label.
Appearance and physical state at room temperature: Yellow-gold liquid.
Storage conditions: Stored in original bottle at 4°C in Building L, Room 109.
Received: April 9, 1992.
Supplier: Best Foods CPC International, Inc. Englewood Cliffs, NJ 07632.
Expiration: April 9, 1993.
Disposition: Retained as vehicle control at SRI in Building L, Room 109.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose Range Finding Assay:
The test article was mixed well in corn oil immediately before dose administration. The volume of the test solution administered was 10 ml/kg of body weight.

Definitive Assay:
The test article was mixed well in corn oil immediately before dose administration. The volume of test suspension administered was 10 ml/kg body weight

Duration of treatment / exposure:

Dose Range Finding Assay:
The preliminary dose range finding assay was performed to determine appropriate dose levels for the definitive study and to define the effects of the test article on the development of erythrocytes in bone marrow. Male and female Swiss-Webster mice were given daily doses of the test article by gavage approximately 24 hours apart for 2 days. Three mice per sex per dose group were individually weighed and dosed with F. Yellow 131 SC in corn oil at dose levels of 300, 600, 1200, 2500, or 5000 mg/kg/day. A control group, consisting of 3 male and 3 female mice, received corn oil at a volume of 10 ml/kg/day. Approximately 48 hours after the final dose administration, all animals were anesthetized by i.p. injection of sodium pentobarbital (60 mg/kg), then euthanized by cervical dislocation.

Definitive Assay:
Mice were dosed by gavage with F. Yellow 131 SC at 1250, 2500, or 5000 mg/kg/day for 2 consecutive days. A corn oil control group, and a benzene positive control group were treated similarly and concurrently with the F. Yellow 131 SC test groups. Half the animals were sacrificed approximately 24 and 48 hours, respectively, after the final dose. Animals were anesthetized by i.p. injection with sodium pentobarbital (60 mg/kg), then euthanized by cervical dislocation.

Frequency of treatment:

Animals in both the range-finding and the definitive assay were dosed once per day.

Post exposure period:

Range-finding Assay: 48 hours
Definitive Assay: Half the animals were sacrificed approximately 24 and 48 hours, respectively, after the final dose.

Dose / conc.:

1 250 mg/kg bw/day

Remarks:

Definitive Assay

Dose / conc.:

2 500 mg/kg bw/day

Remarks:

Definitive Assay

Dose / conc.:

5 000 mg/kg bw/day

Remarks:

Definitive Assay

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: Benzene.
Lot No.: 03825EV.
Purity: 99.9%, as per supplier.
Appearance and physical state at room temperature: Clear, colorless liquid.
Stability: Not available from supplier.
Received: October 10, 1988.
Expiration: October 10, 1998.
Storage conditions: Stored at room temperature in a plastic secondary container at the SRI Chemical Repository, Building M, Room 217.
Supplier: Aldrich Chemical Company P. 0. Box 14508 St. Louis, MO 63178.
Disposition: Retained as positive control at SRI Chemical Repository, Building M, Room 217.

Ten males were dosed with the the positive control at a dose of 500 mg/kg/day.

Tissues and cell types examined:

PERIPHERAL BLOOD ANALYSIS:
Peripheral blood smears from the dose range finding assay were analyzed for the ratio of polychromatic erythrocytes (PCEs) to red blood cells (RBCs).

BONE MARROW ANALYSIS:
Bone marrow smears were analyzed in both the dose range finding and the definitive assays.

Details of tissue and slide preparation:

PERIPHERAL BLOOD ANALYSIS:
Peripheral blood smears from the dose range finding assay were analyzed for the ratio of polychromatic erythrocytes (PCEs) to red blood cells (RBCs). Blood samples were obtained by pricking the ventral tail vein with a 25-gauge needle and drawing 2-3 μ1 of blood into a capillary tube. The sample from each mouse was transferred to 3 clean, prelabeled microscope slides, spread, air-dried, fixed in absolute methanol for 5 minutes, and stored until stained. Slides from each test animal were visually examined, and the slide with the most uniform preparation was coded and stained with acridine orange (Hayashi et al., 1983). Unstained slides were filed for possible future use.

BONE MARROW ANALYSIS:
Bone marrow smears were analyzed in both the dose range finding and the definitive assays. One femur from each mouse was removed and flushed gently with 0.2 ml of fetal bovine serum (FBS) into 0.5 ml of FBS in a 1.5-m1 conical polycarbonate tube. Cells were concentrated by centrifugation, then resuspended in a volume of supernatant, approximately equal to the pellet volumes. The sample from each mouse was transferred to 3 clean, prelabeled microscope slides, spread, air-dried, fixed in absolute methanol for 5 minutes, and stored until stained. Slides from each test animal were visually examined, and the slide with the most uniform preparation was coded and stained with acridine orange (Hayashi et al., 1983). Unstained slides were filed for possible future use.

DATA COLLECTION:
Slides for micronucleus evaluation were coded by an individual not involved in the microscopic evaluation using random-letter codes generated by an SRI-developed software package on an IBM PC program. Slide labels were printed directly from the computer.

CYTOLOGICAL ANALYSIS:
Peripheral blood smears and bone marrow smears were evaluated by using epifluorescence microscopy at a magnification of 630X. In the dose range finding assay, peripheral blood smears and bone marrow smears were analyzed for the number of RNA-positive (polychromatic) erythrocytes (PCEs) in at least 1000 and 200 erythrocytes, respectively, per animal. In the definitive assay, two parameters were determined in the bone marrow smears: (1) the number of micronucleated PCEs in at least 1000 PCEs per animal, which provides an index of chromosomal damage; and (2) the number of PCEs in at least 200 erythrocytes per animal, which provides an index of cytotoxicity.
The criteria used for identifying micronuclei are those described by Schmid (1976), with the additional requirement that the micronuclei exhibit the bright yellow fluorescence characteristic of acridine orange stain. The data from a given slide were
registered directly to an IBM PC data file during scoring. After analysis, the slides were decoded and the data summarized by using a decoding program in an IBM PC.

Evaluation criteria:

DATA ANALYSIS AND INTERPRETATION:
Criteria for a Valid Assay:
The data from this assay were considered acceptable if (1) the frequency of micronucleated cells in the corn oil vehicle control group was within the normal historical range, (2) the administration of the positive control article resulted in a statistically significant elevation of micronucleated cells, and (3) at least 3 surviving animals of each sex in two or more dose groups showed a ratio of PCEs to total erythrocytes in bone marrow greater than or equal to 0.1.

Interpretation of Response:
Positive. The test article is considered positive if the incidence of micronucleated PCEs is significantly higher than that in the corn oil vehicle control group (p < 0.05) in (1) two different dose groups from one experiment, (2) at a single dose if confirmed by a separate experiment (e.g., an increased frequency of micronucleated cells in the dose range finding experiment), or (3) a positive, dose-related increase in the incidence of micronucleated cells.

Negative. The test article is considered negative if the criteria for a positive or inconclusive response are not met.

Inconclusive. The results are considered inconclusive if there is reason to believe that the concentrations of the test article selected for evaluation were inappropriate (e.g., excessive toxicity) or if a statistically significant elevation in the frequency of micronucleated PCEs is observed in only one treatment group and the dose-response trend is not significant.

Control of Bias:
Animals were randomized by using an IBM PC computer-generated, random-number form, then consecutively assigned to dose groups (i.e., animals numbered 1 through 8 were the first 8 animals used in the experiment). Before the microscope slides were scored, they were coded, using random-letter codes, by a person not assigned to score that experiment.

Statistics:

Statistical Tests:
The data were analyzed separately for each sex. The ratio of micronucleated PCEs to PCEs and the ratio of PCEs to total erythrocytes in percentages were calculated for each animal.
The statistical significance of differences in the percentage of PCEs to total erythrocytes among F. Yellow 131 SC-treated and corn oil control groups was evaluated by using the Kruskal-Wallis analysis of variance on ranks (calculated by using an SRI-developed software program in an IBM PC).
In experiments where the frequencies of micronucleated PCEs were determined by scoring 1000 PCEs per animal, data are not expected to be distributed normally. Such data were analyzed by the Cochran-Armitage test for a trend in binomial proportions to determine a significant dose-response relationship (using an SRI-developed software package), and a test for equality of binomial proportions to determine whether values for individual dose groups were statistically different from those for controls (Kastenbaum and Bowman; using an SRI-developed software package). These tests and their rationale are discussed in the ATSM Standard Guide for Conduct of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes and other publications (ATSM Committee, 1988; Margolin et al., 1983).
Animal mean body weights were analyzed by the Student's t-test (in an SRIdeveloped software package) run in an IBM PC.

Sex:

male/female

Genotoxicity:

negative

Remarks:

At doses up to and including 5000 mg/kg/day, F. Yellow 131 SC

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

DOSE RANGE FINDING ASSAY:
For this assay, 3 mice per sex per dose group were individually weighed and dosed orally with F. Yellow 131 SC at 300, 600, 1200, 2500, or 5000 mg/kg/day for 2 consecutive days. A control group, consisting of 3 male and 3 female mice, received corn oil only. Mice were observed daily for four days from the start of dosing until sacrifice.
At 600, 1200, 2500 and 5000 mg/kg/day F. Yellow 131 SC, 1 male mouse, 1 female mouse, 1 male mouse and 1 female mouse, and 2 male mice and 1 female mouse, respectively, had discolored fur due to excretion of the test article. All other mice appeared normal throughout the assay. All mice were sacrificed approximately 48 hours after the last dose administration and cytotoxicity was evaluated by the frequency of RNA-positive erythrocytes (PCEs) among total red blood cells (RBCs).
The number of PCEs among total RBCs was counted in both peripheral blood and bone marrow smears to estimate the frequency of PCEs among erythrocytes. In the peripheral blood, treatment with 300, 600, 1200, 2500, or 5000 mg/kg/day F. Yellow 131 SC produced percentages of PCEs among total RBCs (PCE/RBC ratios) of 1.8, 1.7, 2.3, 2.1, and 1.9% in male mice and 1.5, 1.8, 2.1, 1.6, and 2.6% in female mice, respectively. Treatment with corn oil produced PCE/RBC ratios of 1.9% in male mice and 2.0% in female mice. In bone marrow, treatment with the above doses of F. Yellow 131 SC produced percentages of PCEs among total RBCs (PCE/RBC ratios) of 45, 50, 51, 52, and 50% in male mice and 47, 51, 46, 52, and 51% in female mice, respectively. Treatment with corn oil produced PCE/RBC ratios of 54% in male mice and 47% in female mice. No statistical PCE suppression was observed in either sex in any of the dose groups. No statistical evaluations of the dose range finding data were deemed necessary. The doses selected, in consultation with the Sponsor, for the definitive assay were 1250, 2500, and 5000 mg/kg/day of F. Yellow 131 SC.

DEFINITIVE ASSAY:
For this assay, 10 mice per sex per dose group were individually weighed and dosed orally with F. Yellow 131 SC at 1250, 2500, or 5000 mg/kg/day for 2 consecutive days. A corn oil vehicle control group and a benzene positive control group were treated similarly and evaluated concurrently with the test groups. Five mice per sex per dose group were sacrificed at approximately 24 hours and the rest at approximately 48 hours after the final dose. Mice were observed daily for four days from the start of dosing until death or sacrifice.
Discolored fur due to excretion of F. Yellow 131 SC was observed in 3 male and 1 female mice, 1 male and 2 female mice, and 5 male and 2 female mice at 1250, 2500 and 5000 mg/kg/day, respectively. Rough fur was observed in 1 male mouse treated with benzene, 1 female mouse treated with F. Yellow 131 SC at 2500 mg/kg/day. One male mouse had a humped back and 1 female mouse had discolored fur and rough fur at 5000 mg/kg/day F. Yellow 131 SC. All other mice appeared normal throughout the assay. All mice were evaluated for toxicity and micronucleus formation in bone marrow erythrocytes.
Erythrocytes in bone marrow from treated mice were examined, and the frequency of PCEs among total RBCs and of micronucleated PCEs among PCEs were determined.
At the 24-hour harvest, male and female mice treated with corn oil alone averaged background micronucleus incidences of 0.2 and 0.4%, respectively. F. Yellow 131 SC treated groups had micronucleus counts not statistically different from that of the corn oil vehicle control group.
At the 48-hour harvest, male and female mice treated with corn oil alone averaged background micronucleus incidences of 0.3 and 0.2%, respectively. Again, F. Yellow 131 SC treated groups had micronucleus counts not statistically different from that of the corn oil vehicle control group.
Male mice treated with benzene had micronucleus frequencies approximately 23- and 8-fold greater than that of the corn oil control group at the 24- and 48-hour harvests, respectively. Positive control data were compared with data from the corn oil control group by using binomial proportions, which found statistically significant differences between the treated and control groups at p < 0.01.
It was concluded that F. Yellow 131 SC at doses up to 5000 mg/kg/day did not induce increased incidences of micronuclei in the bone marrow erythrocytes of male and female Swiss-Webster mice. Therefore, F. Yellow 131 SC was considered nongenotoxic under these test conditions.

Conclusions:

In the definitive assay, 10 mice per sex per dose group were dosed orally with F. Yellow 131 SC in corn oil at 1250, 2500, or 5000 mg/kg/day for 2 consecutive days. Five mice per sex per dose group were sacrificed 24 hours after the final dose and the same number 48 hours after the final dose; all mice were evaluated for cytotoxicity and micronucleus formation in bone marrow erythrocytes. A corn oil control group and a benzene positive control group were treated similarly and evaluated concurrently with the test groups.
At doses up to and including 5000 mg/kg/day, F. Yellow 131 SC did not induce increased incidences of micronuclei in the bone marrow erythrocytes of Swiss-Webster mice. Therefore, F. Yellow 131 SC was considered nongenotoxic under these test conditions.

Executive summary:

The genotoxic potential of orally administered Fluorescent Yellow 131 SC (non-volatiles) (F. Yellow 131 SC) to induce micronucleus formation in bone marrow erythrocytes was determined in Swiss-Webster mice.

A dose range finding assay was performed to determine a 2-day maximum tolerated dose (MTD) that would be used to select doses for the definitive study. In this assay, 3 mice per sex received F. Yellow 131 SC in corn oil by oral intubation (gavage) at dose levels of 300, 600, 1200, 2500, or 5000 mg/kg body weight/day (milligrams per kilogram per day). A control group, consisting of 3 male and 3 female mice, received corn oil only. All mice were dosed for 2 consecutive days and were observed daily from the start of dosing until sacrifice. Bone marrow and peripheral blood samples were

obtained approximately 48 hours after the administration of the last dose from all surviving mice. These samples were evaluated for evidence of cytotoxicity reflected in the frequency of RNA-positive (polychromatic) erythrocytes (PCEs) among total erythrocytes (RBCs).

Erythrocytes in both bone marrow and peripheral blood from all mice were examined, and the frequency of PCEs among RBCs was determined. Significant PCE suppression was not observed in either sex in any of the dose groups. No other statistical evaluations were deemed necessary. Therefore, the doses of F. Yellow 131 SC selected for the definitive assay were 1250, 2500, and 5000 mg/kg/day.

In the definitive assay, 10 mice per sex per dose group were dosed orally with F. Yellow 131 SC in corn oil at 1250, 2500, or 5000 mg/kg/day for 2 consecutive days. Five mice per sex per dose group were sacrificed 24 hours after the final dose and the same number 48 hours after the final dose; all mice were evaluated for cytotoxicity and micronucleus formation in bone marrow erythrocytes. A corn oil control group and a benzene positive control group were treated similarly and evaluated concurrently with the test groups.

F. Yellow 131 SC-treated groups had micronucleus counts similar to their respective corn oil control groups both for male and female mice at the 24-and 48-hour harvests.

Male mice treated with benzene had micronucleus frequencies approximately 23-fold greater than those of the corn oil control group at the 24-hour harvest, and approximately 8-fold greater at the 48-hour harvest.

At doses up to and including 5000 mg/kg/day, F. Yellow 131 SC did not induce increased incidences of micronuclei in the bone marrow erythrocytes of Swiss-Webster mice. Therefore, F. Yellow 131 SC was considered nongenotoxic under these test conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial Mutagenicity:

2 assays are available for this substance. In the study performed in 1983 the assay did not include the E.coli WPrvU2 strain. Consequently this assay was repeated more recently in accordance with the current guideline. Both assays were negative for mutagenicity.

Clastogenicity:

1 in vitro assay using rat lymphocytes is available for this substance. The test material was not clastogenic in this assay.

1 in vivo micronucleus assay in mice is available for this substance. The test material did not cause production of micronuclei and thus is concluded not to be clastogenic in vivo. In this assay the test material was a commercial product that contained the REACH substance and some residual solvent. However, the results are consistent with the in vitro study performed using only the REACH substance.

Mammalian cell mutation

1 HGPRT assay is available. The test material was negative for mutagenicity in this assay.

Justification for classification or non-classification

All genotoxicity assays are negative therefore the substance does not require classification for mutagenicity or any further testing.