1,4-bis[[4-(1,1-dimethylethyl)phenyl]amino]-5,8-dihydroxyanthraquinone

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From: September 19, 1997 to October 10, 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Single culture per test conditions instead of triplicate Note: 2-aminoanthracene was used as the sole indicator of the efficacy of the S9-mix. However, each batch was checked prior to its first use for its metabolising capacity by using reference mutagen(s).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Principles of method if other than guideline:

Macrolexgrün G was initially screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. The independent repeat was performed as preincubation for 20 minutes at 37°C. Other conditions remained unchanged.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,4-bis[[4-(1,1-dimethylethyl)phenyl]amino]-5,8-dihydroxyanthraquinone

EC Number:

225-443-9

EC Name:

1,4-bis[[4-(1,1-dimethylethyl)phenyl]amino]-5,8-dihydroxyanthraquinone

Cas Number:

4851-50-7

Molecular formula:

C34H34N2O4

IUPAC Name:

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

Details on test material:

name of test substance: MACROLEX Grün G
Batch n.: 976-011
appearance: dark-green powder
chemical names: 9,10-Anthracenedione, 1,4-bis{[4- (1,1-dimethylethyl)phenyl]amino}- 5,8-dihydroxy
molecular weight: 534
molecular formula: C34H34N2O4
CAS No.: 4851-50-7

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: n/a
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: n/a
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: n/a
- Reactivity of the test material with the incubation material used (e.g. plastic ware): n/a

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): suspended in DMSO
- Preliminary purification step (if any): n/a
- Final concentration of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) n/a

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: n/a

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: made from the livers of at least six adult male Sprague Dawley rats, of approximately 200 to 300 g in weight. For enzyme induction, the animals received a single intraperitoneal injection of Aroclor 1254, dissolved in corn oil, at a dose of 500 mg/kg bw, five days prior to the sacrifice. The animals were prepared unfasted.
The livers were washed with cold (4 °C), 0.15 M KCI solution (approximately 1 ml KCI per 1 g liver), and then homogenized in fresh, cold (4 °C), 0.15 M KCI (approximately 3 ml KCI per 1 g liver). The homogenate was then centrifuged in a cooling centrifuge at 4 °C and 9000 g for 10 minutes. The supernatant (the S9 fraction) was stored at -80 °C in small portions.
- method of preparation of S9 mix:
70 mL of cofactors solution are composed as follows:
MgCl2 x 6 H2O 162.6 mg
KCI 246.0 mg
Glucose-6-phosphate, disodium salt 179.1 mg
NADP, disodium salt 315.0 mg
Phosphate buffer 100.0 mM
The S9 mix comprised 10 % S9 fraction, 70 % cofactor solution and 20 % 0.15 M KCI.
- concentration or volume of S9 mix and S9 in the final culture medium: 10 % S9 fraction
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): Prior to first use, each batch was checked for its metaboliz­ ing capacity by using reference mutagens; appropriate activity was demonstrated. At the beginning of each experiment 4 aliquots of the S9 mix were plated (0.5 ml per plate) in order to assess its sterility. This was repeated after completion of test tube plating. The sterility control plates were then incubated for 48 hours at 37 °C. No in­ dication of contamination of S9 mix was found.

Test concentrations with justification for top dose:

0, 16, 50, 158, 500, 1581 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether (EGDE), and DMF according to information given by the sponsor. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments.
- Justification for percentage of solvent in the final culture medium: not reported.
- Other: MACROLEX Grün G was mixed with DMSO and formed a dark-green suspension.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

Remarks:

No "untreated" negative control was set up for the used solvent, since sufficient evi­ dence was available in the literature (e.g. Maron and Ames, 1983) and from historical control data, indicating that this solvent had no influence.

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

cumene hydroperoxide

other: nitrofurantoin, 4-nitro-1,2-phenylene diamine, 2-amino­ anthracene

Details on test system and experimental conditions:

The initial plate incorporation test followed the directions of Ames. For the mutant count, one plate was used, both with and without S9 mix, for each strain and dose. The independent repeat was performed as preincubation in a water bath at 37°C for minutes. At the end of the preincubation period 2 ml of molten soft agar were added to the tubes, the content mixed and plated.
One plate, filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained one plate per strain. The amount of solvent for the test substance and for the controls was 0.1 ml/plate.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): single
- Number of independent experiments: 2 (plate incorporation and pre-incubation assays)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 0.2ml of thawed culture in 10mL nutrient broth.
- Test substance added: 1/ in agar (plate incorporation) and 2/ pre-incubation

TREATMENT SCHEDULE:
- Preincubation period, if applicable: 20 minutes at 37°C (pre-incubation assay)
- Exposure duration/duration of treatment: 48 hours at 37°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Any supplementary information relevant to cytotoxicity: n/a

- OTHER:n/a

Rationale for test conditions:

In both assays concentrations up to 5000 µg/plate were assessed as per OECD TG 471 (both in the presence and absence of metabolic activation).
At 500 µg/plate the substance started to precipitate.

Evaluation criteria:

The following criteria determined the acceptance of an assay:
a) The negative controls had tobe within the expected range, as defined by published data (e.g. Maron and Ames, 1983) and/ or the laboratories' own historical data.
b) The positive controls had to show sufficient effects, as defined by the laboratories' experience.
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.

Only trials which complied with all three of the above crite­ ria were accepted for assessment. Even if the criteria for points (b) and (c) were not met, a trial was accepted if it showed mutagenic activity of the test compound. Furthermore, an unacceptable trial would have been repeated.

Assessment Criteria
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 150 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to recommended limit concentrations. Precipitation observed from 1581 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to recommended limit concentrations. Precipitation observed from 1581 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to recommended limit concentrations. Precipitation observed from 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to recommended limit concentrations. Precipitation observed from 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to recommended limit concentrations. Precipitation observed from 1581 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not reported
- Data on osmolality: not reported
- Possibility of evaporation from medium: not reported
- Water solubility: not reported
- Precipitation and time of the determination: At 500 µg/plate the substance started to precipitate
- Definition of acceptable cells for analysis: not reported
- Other confounding effects: none

STUDY RESULTS - Table of results are attached to this ESR
- Concurrent vehicle negative and positive control data: The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the sys­tem's sensitivity and the activity of the S9 mix.

Ames test:
- Signs of toxicity: there was no indication of a bacteriotoxic effect at doses of up to and including 5000 pg per plate
- Individual plate counts - cf Table of results
- Mean number of revertant colonies per plate and standard deviation - cf Table of results

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: cf Table of results
- Negative (solvent/vehicle) historical control data: cf Table of results

Applicant's summary and conclusion

Conclusions:

Negative. Evidence of mutagenic activity of MACROLEX Grün G was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

Executive summary:

MACROLEX Grün G was initially screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects, suspended in DMSO, at doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the  histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. The independent repeat was performed as preincubation for 20 minutes at 37°C. Other conditions remained unchanged.

 

Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. Substance precipitation occurred at the dose 500 µg per plate and above.

 

Evidence of mutagenic activity of MACROLEX Grün G was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

 

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide and 2-amino­anthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

 

Therefore, MACROLEX Grün G was considered to be negative (non-mutagenic) without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Despite this sensitivity, no indications of mutagenic effects of MACROLEX  could be found at assessable doses of up to 5000 ug per plate in any of the Salmonella typhimurium strains used.

Due to these results MACROLEX has to be regarded as non-mutagenic.