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Toxicological information

Genetic toxicity: in vitro

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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:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD TG 471and EPA OPPTS 870.5100 and in accordance with the Principles of Good Laboratory Practice (GLP).
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Remarks:
Exceptions related to test substance characterisation - identity, strength, purity and composition, stability of the test substance chemical analyses of the test mixtures and their stability were not performed by the test facility
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Remarks:
same as above
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: dark green crystalline solid
Details on test material:
- Name of test material (as cited in study report): 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ)
- Substance type: dark green crystalline solid
- Physical state: solid
- Analytical purity: > 97% active ingredient
- Lot/batch No.: Lot #020801; TD No. 02-045
- Expiration date of the lot/batch: 31 March 2004
- Stability under test conditions: expected to be stable for the duration of testing
- Storage condition of test material: room temperature

Method

Target gene:
The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs.
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):
- Type and identity of media: Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing -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 shakerlincubator 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 lo9 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system
Test concentrations with justification for top dose:
Preliminary toxicity assay - 3.3, 6.7, 10, 33, 67, 100, 333, 667, 1000 and 1250 µg per plate
Mutagenicity assay - 30, 100, 300,900 and 2500 µg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: Tetrahydrofuran (THF) was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
Details on test system and experimental conditions:
On the day of its use, minimal top agar, containing 0.8 % agar (W/V) and 0.5 % NaCl (W/V), was melted and supplemented with L-histidine, D-biotin and L-tryptophan solution to a final concentration of 50 µM 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, sterile and deionized water produced by the Milli-Q Reagent Water System. Bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar. Nutrient bottom agar was Vogel-Bonner minimal medium E containing 1.5 % (W/V) agar and supplemented with 2.5 % (W/V) Oxoid Nutrient Broth No. 2 (dry powder). Nutrient Broth was Vogel-Bonner salt solution supplemented with 2.5 % (W/V) 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.
One-half (0.5) milliliter of S9 or Sham mix, 100 µL of tester strain and 25 µL of vehicle or test article dilution were added to 2.0 mL of molten selective top agar at 45 ±2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C 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. 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 assay was the preliminary toxicity assay or the plate exhibited toxicity.
Evaluation criteria:
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 was used to conclude a positive result. 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, TAlOO 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:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported

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
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the preliminary toxicity assay, the maximum dose tested was 1250 μg per plate; this dose was achieved using a concentration of 50 mg/mL and a 25 μL plating aliquot. Concentrations from 0.13 to 50 mg/mL were soluble and clear solutions. The dose levels tested were 3.3, 6.7, 10, 33, 67, 100, 333, 667, 1000 and 1250 μg per plate. Precipitate was observed beginning at 10, 33 or 67 μg per plate. No appreciable toxicity was observed.

As a result of the inconsistency in the solubility profile between the solubility test and the preliminary toxicity assay, a second solubility test was conducted. In this test, the test article was definitely insoluble and unworkable at 200 mg/mL, and at least workable at 100 mg/mL. Due to the dark color of the mixture, it wasdifficult to determine if the test article was truly soluble at 100 mg/mL or only workable at this concentration. Based on the findings of the toxicity assay and the
second solubility test, the maximum dose tested in the mutagenicity assay was 2500 μg per plate. This was the highest dose that could be achieved based on the solubility or workability of the test article.

In the mutagenicity assay, no positive mutagenic response was observed. The dose levels tested were 30, 100, 300,900 and 2500 μg per plate. Precipitate was observed beginning at 30, 100 or 300 μg per plate and no appreciable toxicity was observed
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

Under the conditions of this study, test article TBPAAQ [1, 4, 5, 8-Tetra (4'-n-Butylphenylamino) Anthraquinone] was concluded to be negative in the Bacterial Reverse Mutation Assay with an Independent Repeat Assay.
Executive summary:

The test article, TBPAAQ [l,4,5,8-Tetra(4'-n-Butylphenylamino) Anthraquinone], was tested in the Bacterial Reverse Mutation Assay with an Independent Repeat Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA 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 preliminary toxicity assay, was used to establish the dose-range for the mutagenicity assay. The second phase, the mutagenicity assay (initial and independent repeat assays), was used to evaluate the mutagenic potential of the test article.

Tetrahydrofuran (THF) was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. A concentration of 50 mg/mL was a workable suspension.

In the preliminary toxicity assay, the maximum dose tested was 1250 μg per plate; this dose was achieved using a concentration of 50 mg/mL and a 25 μL plating aliquot. Concentrations from 0.13 to 50 mg/mL were soluble and clear solutions. The dose levels tested were 3.3, 6.7, 10, 33, 67, 100, 333, 667, 1000 and 1250 μg per plate. Precipitate was observed beginning at 10, 33 or 67 μg per plate. No appreciable toxicity was observed.

As a result of the inconsistency in the solubility profile between the solubility test and the preliminary toxicity assay, a second solubility test was conducted. In this test, the test article was definitely insoluble and unworkable at 200 mg/mL, and at least workable at 100 mg/mL. Due to the dark color of the mixture, it was difficult to determine if the test article was truly soluble at 100 mg/mL or only workable at this concentration. Based on the findings of the toxicity assay and the second solubility test, the maximum dose tested in the mutagenicity assay was 2500 μg per plate. This was the highest dose that could be achieved based on the solubility or workability of the test article.

In the mutagenicity assay, no positive mutagenic response was observed. The dose levels tested were 30, 100, 300,900 and 2500 μg per plate. Precipitate was observed beginning at 30, 100 or 300 μg per plate and no appreciable toxicity was observed.

Under the conditions of this study, test article TBPAAQ [1, 4, 5, 8-Tetra (4'-n-Butylphenylamino) Anthraquinone] was concluded to be negative in the Bacterial Reverse Mutation Assay with an Independent Repeat Assay