9,10-Anthracenedione, 1,4-bis[(2-ethylhexyl)amino]-5,8-dihydroxy-

Genetic toxicity: in vitro

Administrative data

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:

2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted in compliance with the testing guidelines of the ICH (1996 and 1997) and OECD (1998).

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis[(2-ethylhexyl)amino]-5,8-dihydroxy-9,10-dihydroanthracene-9,10-dione

EC Number:

807-717-5

Cas Number:

295800-70-3

Molecular formula:

C30H42N2O4

IUPAC Name:

1,4-bis[(2-ethylhexyl)amino]-5,8-dihydroxy-9,10-dihydroanthracene-9,10-dione

Test material form:

other: Blue solid

Details on test material:

- Name of test material (as cited in study report): SpectraceTMM D-810 Marker (Solvent Free)
- Physical state: Blue solid
- Lot/batch No.: RWS-2-43
- Expiration date of the lot/batch: 27 September 2007
- Storage condition of test material: Stored at room temperature in the dark.

Method

Target gene:

Specific histidine loci.

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9.

Test concentrations with justification for top dose:

In the confirmatory mutagenicity assay, the dose levels tested were 50, 150, 500, 1500 and 5000 µg per plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: standard solvent

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

Test System:
The tester strains used were the Salmonella typhimuvium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976). Salmonella tester strains were received from Dr. Bruce Ames' designated distributor, Discovery Partners International, San Diego, California. E, coli tester strains were received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).
Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing approximately 50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 37+/-2°C approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of approximately 10E9 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Metabolic Activation System:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mgkg, five days prior to sacrifice. The S9 was batch prepared by BioReliance and stored at -70°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to Salmonella typhimuvium TA100.
The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate, 4 mM 13-nicotinamide-adenine dinucleotide phosphate, 8 mM MgC12 and 33 mM KC1 in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 rnM phosphate buffer at pH 7.4, was prepared immediately before its use. To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar.

Solubility Test:
A solubility test was conducted to select the vehicle. The test was conducted using water, dimethyl sulfoxide (DMSO), ethanol (EtOH) and acetone. The test article was tested to determine the vehicle, selected in order of preference, that permitted preparation of the highest soluble or workable stock concentration, up to 50 mg/mL for aqueous solvents and 500 mg1mL for organic solvents.

Initial Toxicity-Mutation Assay:
The initial toxicity-mutation assay was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. Vehicle control, positive controls and eight dose levels of the test article were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9.

Confirmatory Mutagenicity Assay:
The confirmatory mutagenicity assay was used to evaluate and confirm the mutagenic potential of the test article. Five dose levels of test article along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test article, vehicle control and positive controls were plated in triplicate.

Plating and Scoring Procedures:
The test system was exposed to the test article via the plate incorporation methodology originally described by Arnes et al. (1 975) and updated by Maron and Arnes (1983). On the day of its use, minimal top agar, containing 0.8 % agar (WN) and 0.5 % NaCl (WN), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 pM each. Top agar not used with S9 or Sham mix was supplemented with 25 mL of water for each 100 mL of minimal top agar. For the preparation of media and reagents, all references to water imply sterile, deionized water produced by the Milli-Q Reagent Water System. Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5 % (WIV) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (WN) agar and supplemented with 2.5 % (WIV) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (WN) Oxoid Nutrient Broth No. 2 (dry powder).
Each plate was labeled with a code system that identified the test article, test phase, dose level, tester strain and activation, as described in detail in BioReliance's Standard Operating Procedures.
One-half (0.5) milliliter of S9 or Sham mix, 100 yL of tester strain and 50 yL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45+2OC. After vortexing, the mixture was overlaid onto the surface of 25 rnL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 yL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37*2OC. Plates that were not counted immediately following the incubation period were stored at 2-8OC until colony counting could be conducted.
The condition of the bacterial background lawn was evaluated for evidence of test article toxicity by using a dissecting microscope. Precipitate was evaluated by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the Codes for Scoring Toxicity and Degree of Precipitation table.
Revertant colonies for a given tester strain and activation condition, except for positive controls, were counted either entirely by automated colony counter or entirely by hand unless the plate exhibited toxicity.

Evaluation criteria:

Evaluation of Results:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article. Data sets for tester strains TA1535 and TA1537 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 vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA 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 vehicle control value.

Statistics:

not applicable

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Solubility Test:
Dimethyl sulfoxide (DMSO) was selected as the solvent based on solubility of the test article and compatibility with the target cells. The test article formed workable suspensions in dimethyl sulfoxide from 50 to 100 mglmL in the solubility determination.

Sterility Results:
No contaminant colonies were observed on the sterility plates for the vehicle control, the test article dilutions and the S9 and Sham mixes.

Initial Toxicity-Mutation Assay:
In the initial toxicity-mutation assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 µL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. The test article formed
workable suspensions in DMSO from 1.0 to 100 mg/mL and soluble and clear solutions from 0.030 to 0.30 mg/mL. In the initial toxicity-mutation assay, no positive mutagenic response was observed. Precipitate was observed beginning at 50 µg per plate. No appreciable toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 µg per plate.
In Experiment B1 (Initial Toxicity-Mutation Assay), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

None.

Applicant's summary and conclusion

Conclusions:

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

All criteria for a valid study were met. The results of the bacterial reverse mutation assay indicate that, under the conditions of ths study, Spectrace MD-810 Marker (Solvent Free) did not cause a positive response in either the presence or absence of Aroclor-induced rat liver S9.

Executive summary:

The test article, Spectrace® MD-810 Marker (Solvent Free), was tested in the bacterial reverse mutation assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2uvrA in the presence and absence of Aroclor-induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article.

Dimethyl sulfoxide (DMSO) was selected as the solvent based on solubility of the test article and compatibility with the target cells. The test article formed workable suspensions in dimethyl sulfoxide from 50 to 100 mg/rnL in the solubility determination.

In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. The test article formed workable suspensions in DMSO from 1.0 to 100 mg/mL and soluble and clear solutions from 0.030 to 0.30 mg/mL. In the initial toxicity-mutation assay, no positive mutagenic response was observed. Precipitate was observed beginning at 50 μg per plate. No appreciable toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.

In the confirmatory mutagenicity assay, no positive mutagenic response was observed. The dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed beginning at 50 or 150 μg per plate. No appreciable toxicity was observed.

Under the conditions of this study, test article Spectrace® MD-810 Marker (Solvent Free) was negative in the bacterial reverse mutation assay (Ames test).