2,20-dichloro-13,31-diethyl-4,22-dioxa-13,18,31,36-tetraazanonacyclo[19.15.0.03,19.05,17.06,14.07,12.023,25.024,32.025,30]hexatriaconta-1(36),2,5,7,9,11,14,16,18,20,23,25,27,29,32,34-hexadecaene

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed in compliance with GLP and OECD Test guideline 471

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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2,20-dichloro-13,31-diethyl-4,22-dioxa-13,18,31,36-tetraazanonacyclo[19.15.0.0³,¹⁹.0⁵,¹⁷.0⁶,¹⁴.0⁷,¹².0²³,³⁵.0²⁴,³².0²⁵,³⁰]hexatriaconta-1(36),2,5,7(12),8,10,14,16,18,20,23(35),24(32),25,27,29,33-hexadecaene

EC Number:

606-790-9

Cas Number:

215247-95-3

Molecular formula:

C34H22Cl2N4O2

IUPAC Name:

2,20-dichloro-13,31-diethyl-4,22-dioxa-13,18,31,36-tetraazanonacyclo[19.15.0.0³,¹⁹.0⁵,¹⁷.0⁶,¹⁴.0⁷,¹².0²³,³⁵.0²⁴,³².0²⁵,³⁰]hexatriaconta-1(36),2,5,7(12),8,10,14,16,18,20,23(35),24(32),25,27,29,33-hexadecaene

Test material form:

solid: nanoform

Method

Target gene:

his- for S. typhimurium strains
trp- for E. coli WP2 uvr A

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (phenobarbital/ß-naphthoflavone-induced rat liver S9)

Test concentrations with justification for top dose:

Experiment I (plate incorporation): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without metabolic activation
Experiment II (pre-incubation): 33, 100, 333, 1000, 2500 and 5000 µg/plate both with and without metabolic activation

Vehicle / solvent:

DMSO, purity >99% (Merck, Darmstadt, Germany). The solvent was chosen because of its solubility properties and its relative non-toxicity to bacteria

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below for additional information

Details on test system and experimental conditions:

METHOD OF APPLICATION:
plate incorporation (experiment I); preincubation (experiment II). Since experiment I gave a negative result, experiment II was performed as a
preincubation assay.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY: existence of evaluable plates (> 0 colonies) at five concentrations or more

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

Not mandatory according to OECD guideline 471

Results and discussion

Test results

Test results 1

Key result

Species / strain:

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

Metabolic activation:

with

Genotoxicity:

negative

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

Test results 2

Key result

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

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

Test results 3

Key result

Species / strain:

E. coli WP2 uvr A

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:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Pre-experiment was reported as experiment I because the criterion (evaluable plates (>0 colonies) at five concentrations or more in all strains are used) was met.

COMPARISON WITH HISTORICAL CONTROL DATA:
yes, see below

ADDITIONAL INFORMATION ON CYTOTOXICITY:
see below

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in both experiments.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix) with the exception of strain TA 98 without metabolic activation in experiment II. This strain showed a minor increase in revertant colony numbers at all concentrations of the test item. The absolute numbers of colonies reached and exceeded the threshold of two times the number of the corresponding solvent control at 33 and 100 μg/plate. To verify the results of this experiment an independent repeat experiment was performed under identical conditions with strain TA 98 in the absence of metabolic activation. No increase in the number of revertant colonies occurred in the repeat experiment and the effect observed in the second experiment was judged as biologically irrelevant. The repeat experiment is reported as experiment II A (see below).

There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In experiment I, the data in the negative and solvent control of strain WP2 uvrA were slightly above our historical control range in the presence and absence of metabolic activation. The number of colonies did not quite reach the lower limit of our historical control range in the solvent control of strain TA 98 without S9 mix in experiment II. Since these deviations are rather small, these effects are considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

In strains TA 1535 and WP2 uvrA of the first experiment with metabolic activation the historical range of positive controls was just not reached (200 versus 221 colonies). This minor effect was judged to represent fluctuations. The threshold of two times or three times the corresponding solvent control was exceeded (factor of 5.3 and 2.5), so the test was considered valid.

Exp. II: preincubation method without S9 mix

Concentrations given in µg/plate

Strain -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA98 -- 2.1 -- 2.0 -- 1.9 -- 1.7 -- 1.9 -- 1.2

Exp. IIa: preincubation method without S9 mix (repeat assay)

Concentrations given in µg/plate

Strain -- 33 -- 100 -- 333 -- 1000 -- 2500 -- 5000

TA98 -- 1.0 -- 0.6 -- 0.6 -- 0.7 -- 1.0 -- 0.6

Applicant's summary and conclusion

Conclusions:

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

The test item (Pigment Violet 23) did not induce gene mutations by frameshifts or base-pair substitutions in the genome of the strains used when tested in a bacterial reverse mutation assay (Ames test, test strains: Salmonella typhimurium TA 98, TA 100, TA 1535, TA1537; E. coli WP2 uvrA) with and without metabolic activation (rat liver S9 and hamster liver S9) at up to 5000 µg/plate. Since the test item did not induce gene mutations Pigment Violet 23 is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of Pigment Violet 23 to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: Pre-Experiment and Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Hostaperm-Violett RL spez at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.