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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

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)
Principles of method if other than guideline:
MACROLEX Violett 3R 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:
no
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1,8-bis[(4-methylphenyl)amino]anthraquinone
EC Number:
201-398-0
EC Name:
1,8-bis[(4-methylphenyl)amino]anthraquinone
Cas Number:
82-16-6
Molecular formula:
C28H22N2O2
IUPAC Name:
1,8-bis[(4-methylphenyl)amino]-9,10-dihydroanthracene-9,10-dione
Specific details on test material used for the study:
name of test substance: MACROLEX Violett 3R BAYER AG
appearance: violet powder
chemical names: 9,10-Anthracenedione, 1,8-bis[(4- methylphenyl)amino]-
molecular weight: 418
molecular formula: C28H22N2O2
CAS No.: 82-16-6

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:
S9 mix
Test concentrations with justification for top dose:
The following doses per plate were evaluated [µg per plate]:
Negative control 0
MACROLEX Violett 3R 5000
MACROLEX Violett 3R 1581
MACROLEX Violett 3R 500
MACROLEX Violett 3R 158
MACROLEX Violett 3R 50
MACROLEX Violett 3R 16
Vehicle / solvent:
MACROLEX Violett 3R was mixed with DMSO and formed a violet suspension.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
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.


The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 µg or 5 µl per plate were used as the highest dose. At least five additional doses were routinely used. If less than three doses were used for assessment, at least two repeats were performed. The results of the first experiment were then considered as a pre-test for toxicity. However, in case of a positive response or if at least three doses could be used for assessment, the first trial was included in the assessment. If the second test confirmed the results of the first, no additional repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first experiment.

The independent repeat was performed as preincubation in a water bath at 37°C for 20 minutes. At the end of the preincubation period 2 ml of molten soft agar were added to the tubes, the content mixed and plated.

For the mutant count, one plate was used for each strain and dose. One plate, filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained one plate per strain. In experiments without S9 mix buffer was used as replacement.

The doses of this trial were determined on the basis of the results of the plate incorporation assay. Doses are given as µg/tube for better separation of plate incorporation and preincubation trials, despite the fact that µg/plate and µg/tube could be used synonymously.

The bacterial suspensions were obtained from 17-hour cultures in nutrient broth, which had been incubated at 37°C and 90 rpm. These suspensions were used for the determination of mutant counts. No standardized procedure was employed to set the bacterial suspensions at a defined density of viable cells per milliliter, since the chosen method of incubation normally produces the desired density. However, the numbers of viable cells were established in a parallel procedure by determining the titers.

The dilution of bacterial suspensions used for the determination of titers was 1:1,000,000. Titers were determined under the same conditions as were the mutations, except that the histidine concentration in the soft agar was increased five­ fold to permit the complete growth of bacteria. The tests were performed both with and without 89 mix.
The count was made after the plates had been incubated for 48 hours at 37°C. If no immediate count was possible, plates were temporarily stored in a refrigerator.




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
Key result
Species / strain:
S. typhimurium, other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
There was no indication of a bacteriotoxic effect of MACROLEX Violett 3R at doses of up to and including 5000 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. At 158 µg per plate, the substance started to precipitate.

None of the five strains concerned showed in the plate incorporation part a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the preincubation trials.

Applicant's summary and conclusion

Conclusions:
Negative. Evidence of mutagenic activity of MACROLEX Violett 3R was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.
Executive summary:

MACROLEX Violett 3R 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.

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 158 µg per plate and above.

Evidence of mutagenic activity of MACROLEX Violett 3R 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 Violett 3R was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/ microsome test.