Reaction mass of bis[3-[[4-[benzylmethylamino]phenyl]azo]-1,4-dimethyl-1H-1,2,4-triazolium] tetrachlorozincate(2-) and bis[5-[[4-[benzylmethylamino]phenyl]azo]-1,4-dimethyl-1H-1,2,4-triazolium] tetrachlorozincate(2-)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was found to be mutagenic in bacteria tester strains S. typhimurium TA 98 and TA 1538 in the presence of an exogenous metabolic activation system at cytotoxic concentrations. The substance was not found to be genotoxic in the in vitro chromosome aberration test. No point mutations were found in the assay with V79 chinese hamster lung fibroblasts neither in presence nor absence of an exogenous metabolic activation system.

The substance was not found to be genotoxic in the in vitro HPRT assay.

No clastogenic effect was observed in the assay with V79 chinese hamster lung fibroblasts neither in presence nor absence of an exogenous metabolic activation system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May 11, 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0.2, 2, 20, 200 and 2000 µg per Petri dish.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: daunomycine

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-anthramine

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar.
The bacterial strains were kept as frozen broth cultures, in aliquots of 0.5 ml at -70 °C with 8.0 % dimethylsulfoxide (DMSO). Fresh cultures were prepared by adding 0.1 ml of a thawed stock culture to 15 ml of nutrient broth (normal Difco nutrient broth for strains TA 1535 and TA 100, and double strength Difco nutrient broth for TA 1537 and TA 98, in 0.5 % NaCI). A nutrient agar (2.5% Difco nutrient broth, 1.2 % Difco agar) was also streaked. The broth was incubated in the dark in a shaking water bath at 37 °C for 16 hours, while the plate was incubated at 37 °C overnight. Broths and plates were then transferred to the refrigerator, for up to one week.

NUMBER OF REPLICATIONS:3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 200 µg only

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 100 and TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 1535, TA 1537 and TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 1537 and TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, product was found to be mutagenic for S. typhimurium strain TA 98 in the presence of a liver microsomal enzyme preparation (S9-mix) from male rats pretreated with Aroclor 1254. The evidence for mutagenicity existed at a a concentration level of 200 µg of product per Petri dish, at 2000 µg the decrease of the number of revertants is due to a toxic effect as well as the absence of the background "lawn" in strains TA 1535 and TA 100 at the highest concentration (2000 µg) without metabolic activation.

Conclusions:

The test substance was found to be mutagenic for S. typhimurium strain TA 98 in the presence of a liver microsomal enzyme preparation (S9-mix) at cytotoxic concentratons.
A toxic effect in strains TA 1535 and TA 100 at the highest concentration (2000 µg) without metabolic activation was observed.

Executive summary:

The substance was tested according to the OECD Guideline 471 with the Salmonella typhimurium strains TA I535, TA I537, TA 98 and TA l00 at concentrations from 0.2 to 2000 µg per Petri dish both in the presence and absence of metabolic activation.

Under the test condition the test substance was found to be mutagenic for S. typhimurium strain TA 98 in the presence of a liver microsomal enzyme preparation (S9-mix) at cytotoxic concentrations. Moreover a toxic effect in strains TA 1535 and TA 100 at the highest concentration (2000 µg) without metabolic activation was observed. Based on the results observed the substance, under the test conditions, is considered as mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

January 15, 1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium, other: TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

100, 200, 400, 800, 1600 and 3200 µg/0.1 ml

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

cyclophosphamide

other: daunorubicin-Hcl,

Details on test system and experimental conditions:

Number of replications: 3

Species / strain:

S. typhimurium, other: TA98 and TA1538

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 400, 800, 1600 µg/0.1 mL only

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 100, TA 1535, TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 100 and TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA 98, TA 1537, TA 1538

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

In the experiments performed without microsomal activation comparison of the number of histidine-prototrophic mutants in the

controls and after treatment with test item revealed a reduction in the colony count due to a growth-inhibiting effect of the compound at the concentration of 3200 µg/0.1 ml.

In the experiments performed with microsomal activation, treatment with test item led to an increase in the number of backmutant

colonies of strains TA 98 and TA 1538. A mutagenic effect was observed at the concentrations of 400, 800 and 1600 µg/0.1 ml.

Conclusions:

The test substance was found to be mutagenic for S. typhimurium strain TA 98 and TA 1538 in the presence of a liver microsomal enzyme preparation (S9-mix) at cytotoxic concentrations. A toxic effect in strains TA 1535 and TA 100 at the highest concentration (2000 µg) without metabolic activation was observed at the concentration of 3200 µg/0.1 ml.

Executive summary:

The substance was tested according to the OECD 471 for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium with the strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 100, 200, 400, 800, 1600 and 3200 µg/0.1 ml.

In order to confirm the results, the experiments on Strains TA 98 and TA 1538 were repeated. These tests permit the detection of point mutations in bacteria induced by chemical substances.

Results

In the experiments performed without microsomal activation on Strains TA 100 and TA 1535, comparison of the numbers of backmutant colonies in the controls and the cultures treated with the various concentrations of test item revealed a reduction in the colony count due to a growth inhibiting effect of the compound at the concentration of 3200 µg/0.1 ml.

In the experiment performed with microsomal activation on Strains TA 98 and TA 1538, treatment with test substance led to an increase in the number of back-mutant colonies accompanied by cytotoxicity, at the concentrations of 400, 800 and 1600 µg/0.1 ml.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From March 26, 1985 to May 9, 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Principles of method if other than guideline:

In vitro test for mutagenic properties of substances in mammalian cells with and without microsomal activation.
Evaluation of any property of the test substance or its metabolites to induce point mutations.

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 Chinese hamster cells, originally derived from embryonic lung tissue, obtained from Dr. D.Wild, Freiburg, FRG.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

without metabolic activation 0.075 µg/ml, 0.15 µg/ml, 0.30 µg/ml, 0.60 µg/ml, 1.20 µg/ml, 1.80 µg/ml, 2.40 µg/ml, 3.00 µg/ml
with metabolic activation 0.50 µg/ml, 1.00 µg/ml, 2.00 µg/ml, 4.00 µg/ml, 8.00 µg/ml, 12.00 µg/ml, 16.00 µg/ml, 20.00 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

with and without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

NUMBER OF REPLICATIONS:8

NUMBER OF CELLS EVALUATED:250000 cells

CYTOTOXICITY
A cytotoxicity test was performed on V79 cells as a preliminary test to determine the highest concentration of the test substance to be applied in the mutagenicity assay. The treatment schedule was identical to that applied later in the mutagenicity study (5-hour treatment in the experiment with activation and 21 hour in the experiment without activation) . The concentration to be selected as the highest for the mutagenicity assay is that causing about 90 % reduction of viable cells in the cytotoxicity test in comparison with the negative (medium) control. To each dish, 200 V79 cells were seeded in growth medium (Ham's F10, supplemented with 10% foetal calf serum and antibiotics). On the following day, the cells were exposed to the test substance either for five hours in the presence of the activation mixture or for twenty-one hours in the absence of the activation mixture. In the two parts of the experiment, 17 concentrations of the substance were applied and one solvent control. The highest concentration tested was 10.0 mg/ml; the lower concentrations decreased by a factor of 0.5. Consequently, the concentrations in both parts of the cytotoxicity test ranged from 153 ng/ml to 10.0 mg/ml. The treatment was terminated by washing the cultures with buffer, followed by reincubation in fresh growth medium for a further six days. Thereafter, the cultures were fixed and stained with Giemsa and the surviving colonies determined with the aid of an electronic colony counter (Fisher Count-All TM , model 600).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The substance and its metabolites did not induce point mutation in mammalian cells.

Executive summary:

The substance was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The experiments were performed without

microsomal activation at concentrations of 0.075, 0.15, 0.30, 0.60, 1.20, 1.80, 2.40 and 3.00 µg/ml and with microsomal

activation at concentrations of 0.50, 1.00, 2.00, 4.00, 8.00, 12.00, 16.00 and 20.00 µg/ml. This system allows the detection of point mutations, frameshift mutations and deletions induced by the test substance or by its metabolites. Mutagenic effects are manifested

by the appearance of cells resistant to 6-thioguanine (6-TG) or 8-azaguanine (8-AG) and can be quantified by comparison of the numbers of 6-TG- or 8-AG- resistant colonies in the treated and control cultures. To ensure that any mutagenic effect of metabolites of the test substance found in mammals are also detected, a parallel series of experiments is performed, in which its metabolic turnover is simulated in vitro by the addition of an activation mixture containing rat liver microsomes and co-factors to the cell

cultures.

In the experiment performed without microsomal activation, comparison of the numbers of mutant colonies in the controls

and the cultures treated with the various concentrations revealed no significant deviations, either in these cultures screened with 6-TG or in those screened with 8-AG. Comparison of the mutant frequency in the control and the treated cultures gave factors of 1.0 at all concentrations tested and screened with 6-AG. In the cultures screened with 6 -TG factors of 1.0 (at 0.075 µg/ml, 0.30 µg/ml and

1.80 µg/ml), 1.1 (at 0.60 µg /ml) , 1.3 (at 0.60 µg/ml) , 2.0 (at 1.20 µg/ml) and 2.2 (at 0.15 µg/ml and 3.00 µg/ml) were observed .

In the experiment performed with microsomal activation, comparison of the number of mutant colonies in the controls and in the cultures treated with various concentratons of the test substance revealed no significant deviation, either in these cultures treated with 8-AG or in those screened with 6-TG.

The two higest concentrations tested were too toxic and the cultures could not be subjected to the mutant selection procedure.

Comparison of the mutant frequency in the control and the treated cultures gave factors of 1.0 (at 0.5 µg/ml, 1.0 µg/ml, 2.0 µg/ml and 4.0 µg/ml) and 1.2 (at 8.0 µg/ml) and 1.5 (at 12.0 µg/ml) in the cultures screened with 8 -AG.

In the cultures screened with 6-TG factors of 1.0 (at 0.5 µg/ml, 1.0 µg/ml, 2.0 ug/ml, 4.0 µg/ml and 12.0 µg/ml) and 2.6 (at 8.0 µg/ml) were obtained.

All enhancements are regarded as not significant, because the corresponding mutant frequencies are in the range of the historical values obtained from negative controls. Thus, in the investigations performed without and with microsomal activation, a mutant factor greater than 3.0 together with a difference in the treated and untreated dishes of at least 20 clones per 10^6 cells plated was not detectable at any concentration tested, and there was also no indication, based of significantly enhanced mutant frequency values, of

a concentration-mutant frequency relation in either experiment. Comparison of the concurrent "positive controls" with EMS

(300 nl/ml) and the negative controls in the experiment without microsomal activation gave factors of 287.9 (selection

with 8-AG) and 463.8 (selection with 6-TG). In the experiment with metabolic activation, comparison of the mutant frequency values in DMN (1 ul/ml)-treated positive controls and negative controls revealed factors of 33.8 (selection with 8-AG) and 44.8 (selection with 6-TG).

It is concluded, that under the given experimental conditions the substance and its metabolites induced no mutagenic effects in this forward mutation system.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From October 07, 1998 to November 12, 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

The study was performed as a screening test in one experiment with a single preparation interval (18 h) and evaluation of the highest possible test article concentration.

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

The study was performed as a screening test in one experiment with a single preparation interval (18 h) and evaluation of the highest possible test article concentration.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The cells have a stable karyotype with a modal chromosome number of 22.
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, D-64287 Darmstadt) were stored in liquid nitrogen in the cell bank of RCC-CCR allowing the repeated use of the same cell culture batch in experiments. Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

without S9 mix: 1.25, 2.5, 5.0, 10, 20 µg/ml
with S9 mix: 1.875, 3.75, 7.5, 15, 30 µg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium. Minimal Essential Medium.

DURATION
- Preincubation period:
- Exposure duration:
- Expression time (cells in growth medium):
- Selection time (if incubation with a selection agent):
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays):
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays):

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 41000 cells/slide

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
Pre-Test on Toxicity
A pre-test on cell growth inhibition with 4 h (with S9 mix) and 24 h (without S9 mix) treatment was performed in order to determine the toxicity of the test article.
Cytotoxicity was determined using concentrations separated by no more than a factor of 2 - 3.33. The general experimental conditions in this pre-test were the same as described below for the cytogenetic main experiment. The following method was used:
In a quantitative assessment, exponentially growing cell (seeding 41000 cells/slide, with regard to the culture time 48 h) were treated with the test article for simulating the conditions of the main experiment. A qualitative evaluation of cell number and cell morphology was made 4 h after start of treatment. 24 h after start of treatment the cells were stained and in 10 coordinate defined fields of the slides (2 slides per treatment group)
the cells were counted. The cell number of the treatment groups is given as % cells in relation to the control.

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Evaluation criteria:

A test article is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- no significant increase of the number of structural chromosome aberrations are observed.

A test article is classified as mutagenic if:
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.0 % aberrant cells exclusive gaps).
- either a concentration-related or a significant increase of the number of structural chromosome aberrations are observed.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test (10) (p < 0.05).
However, both biological and statistical significance should be considered together.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No influence of the test article on the pH value or osmolarity was observed
- Precipitation: In the pre-test, precipitation of the test article in culture medium was observed at 850 µg/ml and above in the absence and at 106.3 µg/ml and above in the presence of S9 mix.

RANGE-FINDING/SCREENING STUDIES: was performed.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The cytogenetic evaluation of higher concentrations in the respective intervals (with and without S9 mix) was impossible due to strong test article induced toxic (low metaphase numbers < 10 % of control).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The substance was not found to be mutagenic in this in vitro chromosome aberration test with V79 cells.

Executive summary:

In this screening study, the test article, dissolved in culture medium (MEM), was assessed for its potential to induce structural chromosome aberrations in the V79 cell line of the Chinese hamster using the following study design.

	without S9-mix	with S9-mix
Exposure period	18 h	4h
Recovery	-	14 h
Preparation interval	18 h	18 h

In the absence and the presence of S9 mix the highest possible test article concentrations were evaluated. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest concentration used in a pre-test on toxicity (3400 µg/ml) was chosen with regard to the current OECD-Guideline for in vitro mammalian cytogenetic tests. Dose selection of the cytogenetic experiments was performed considering the toxicity data of the pre-test. The chosen treatment concentrations and the rational are described on page 15. In this study no influence of the test article on the pH value or osmolarity was observed.

In the main experiment, reduced cell numbers were observed in the absence of S9 mix after continuous treatment with 5.0 µg/ml. In the presence of S9 mix after 4 h treatment no clear toxicity was observed at the highest evaluated concentration. Neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test article.

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

Conclusion

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in

vitro.

Therefore, the substance is considered to be non mutagenic in this chromosome aberration test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Regarding mutagenic effects in bacterial cells, there was only a weak mutagenic action on strains S. typhimurium TA 98 and TA 1538 observed, when administered at cytotoxic concentrations. Whereas, the in vitro gene mutation study in mammalian cells and the in vitro cytogenicity/chromosome aberration study in mammalian cells did not result in any concerns regarding mutagenicity and/or clastogenicity.

 

Data available on Basic Red 46 showed that the test substance is orally bioavailable but does not penetrate through the skin. This is shown by an oral LD50 of the active ingredient of 560 mg/kg bw in male and female rats and a dermal LD50 of > 2000 mg/kg bw in male and female rabbits.

A combined repeat-dose/developmental screening study by oral gavave in male and females rats was carried out at dose levels of 25, 75, and 175 mg/kg bw/day and did not result in any significant toxicological relevant effects. The ‘No Observed Adverse Effect Level’ (NOAEL) for systemic and reproductive toxicity was therefore considered to be 175 mg/kg bw/day for either sex. During necropsy and histopathology no staining of organs and tissues was observed, showing that the test substance did not distribute into organs. Due to its molecular weight is it expected that the majority of the substance is excreted via faeces, however, the staining of the bedding showed that a distinct amount of the absorbed substance is excreted via kidney/urine. Unabsorbed test material is expected to be excreted with the faeces.

While staining of skin and fur has been observed, no indication has been provided that the test material is able to pass internal tissue barriers like the blood-brain barrier, the blood-placenta barrier or the blood-testis barrier. In addition, the histopathological report confirmed, that there were no test item related microscopic findings in reproductive organs. The only histopathological finding related to treatment was due to irritating effects of the test substance on the non-glandular stomach in high dose animals and mid dose males. The toxicokinetic assessment performed on this test substance confirmed that based on the available study data it is reasonable to assume that Basic Red 46 would in all probability be absorbedviathe gastrointestinal tract subsequently entering the circulatory system in the blood. For any test item that is absorbed following oral ingestion, the high water solubility should facilitate the distribution of the test substance throughout the body in the water compartment of circulatory systems. The chemical structure of the test item, with its lack of significant reactive groups on the molecule suggest that the test item will not bind to proteins within the circulatory system. Accumulation of the test substance in body fat is not expected due to the high water solubility and low octanol:water partition coefficient value. The passage of test item across specialized biological membranes such as the blood/brain, blood/testis or blood/placental barrier will be subject to the same physical limitations (high molecular weight and low octanol:water partition coefficient) as with absorption from the gastro-intestinal tract and may therefore suggest that biological distribution will be inhibited across such biological structures. This suggestion may be supported by the lack of test item stained cellular structures such as brain and testis identified from histopathological assessment of treated animals from the repeated dose toxicity study.

Consequently, it is highly unlikely that the test substance may reach gonadal cells, enter these cells through the cell wall, enter the cell core and exert any hereditable mutagenic effects. In addition, Basic Red 46 has demonstrated to exert no mutagenic or cytogenic behaviour when tested on mammalian cells in vitro when administered at concentration far exceeding concentrations that are achievable in in vivo studies.

 

Due to these facts, the positive effects seen in S. typhimurium strains TA 98 and TA 1538 are considered incidental effects due to cytotoxicity interfering with the proper conduct of the assay, leading to false positive results.

As the Ames test is an overly sensitive test and the test in bacteria tends to lead to false positive results with respect to mammalian species, the in vitro mammalian cell gene mutation test (HPRT-assay) was added to the test battery, to assess the mutagenic properties of the test substance in a more relevant test system for humans, following the “Guidance on a strategy for genotoxicity testing of chemicals substances” (COM 2011) as it was reported to be the test in mammalian cells to provide more reliable test results (Glatt 1994, Kirkland 2005 and 2011). It covers the same endpoint – mutagenicity – as the bacterial reverse mutation assay but is conducted in the more relevant cell system – mammalian cells. Where the mutation assays (in both, bacterial and mammalian cells), detectgene mutationssuch as base pair substitutions, frameshifts, small deletions and insertions; the in vitro chromosome aberration using mammalian cultured cell (OECD 473), identifies substances that causestructural chromosomal aberrations(in the chromosome or chromatid), such as structural breaks, gaps, exchanges, polyploidy or endoreduplication and adds even more information on the effects of the test substance on the whole chromosome.

 

As, literature data show that almost all Ames-positive rodent carcinogens are also positive in at least one of thein vitromammalian cell tests(Kirkland 2011 and 2014), the assumption that this test result is a false positive result due to cytotoxic effects is confirmed by the two negative results in mammalian cells.

 

COM 2011. A Strategy for Testing of Chemicals for Genotoxicity. Committee on Mutagenicity of Chemicals in Food, Consumer Products and the Environment (COM). September 2011

Glatt (1994). Comparison of common gene mutation tests in mammalian cells in culture: a position paper of the GUM Commission for the Development of Guidelines for Genotoxicity Testing. Mutat Res. 1994;313(1):7–20

Kirkland et al (2005). Evaluation of the ability of a battery of three in-vitro genotoxicity tests to discriminate rodent carcinogens and non-carcinogens. I. Sensitivity, specificity and relative predictivity.Mutat Res. 2005 July 4;584(1–2):1–256

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Additional information

The substance was tested according to the OECD 471 for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium with the strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100. These tests permit the detection of point mutations in bacteria induced by chemical substances. The investigations were performed with the following concentrations with and without microsomal activation: 100, 200, 400, 800, 1600 and 3200 µg/0.1 ml. To confirm the results, the experiments on strains TA 98 and TA 1538 were repeated. In the experiments performed without microsomal activation on Strains TA 100 and TA 1535, comparison of the numbers of revertant colonies in the controls and the cultures treated with the various concentrations of test item revealed a reduction in the colony count due to a growth inhibiting effect of the compound at the concentration of 3200 µg/0.1 ml. In the experiment performed with microsomal activation on strains TA 98 and TA 1538, treatment with test substance led to an increase in the number of revertant colonies at the concentrations of 400, 800 and 1600 µg/0.1 ml in the presence of cytotoxic effects.

 

In a second test, the substance was tested according to the OECD Guideline 471 with the Salmonella typhimurium strains TA I535, TA I537, TA 98 and TA l00 at concentrations from 0.2 to 2000 µg per Petri dish both in the presence and absence of metabolic activation. Under the test condition the test substance was found to be mutagenic for S. typhimurium strain TA 98 in the presence of a liver microsomal enzyme preparation (S9-mix) at the dose level of 200 µg/plate only, in the presence of cytotoxic effects. Moreover, a toxic effect in strains TA 1535 and TA 100 at the highest concentration (2000 µg) without metabolic activation was observed.

 

 

Basic Red 46 was tested for mutagenic effects on V79 Chinese hamster cells in vitro. The experiments were performed without microsomal activation at concentrations of 0.075, 0.15, 0.30, 0.60, 1.20, 1.80, 2.40 and 3.00 µg/ml and with microsomal activation at concentrations of 0.50, 1.00, 2.00, 4.00, 8.00, 12.00, 16.00 and 20.00 µg/ml. This system allows the detection of point mutations, frameshift mutations and deletions induced by the test substance or by its metabolites. Mutagenic effects are manifested by the appearance of cells resistant to 6-thioguanine (6-TG) or 8-azaguanine (8-AG) and can be quantified by comparison of the numbers of 6-TG- or 8-AG- resistant colonies in the treated and control cultures. To ensure that any mutagenic effect of metabolites of the test substance found in mammals are also detected, a parallel series of experiments is performed, in which its metabolic turnover is simulated in vitro by the addition of an activation mixture containing rat liver microsomes and co-factors to the cell cultures.

In the experiment performed without microsomal activation, comparison of the numbers of mutant colonies in the controls and the cultures treated with the various concentrations revealed no significant deviations, either in these cultures screened with 6-TG or in those screened with 8-AG. Comparison of the mutant frequency in the control and the treated cultures gave factors of 1.0 at all concentrations tested and screened with 6-AG. In the cultures screened with 6 -TG factors of 1.0 (at 0.075 µg/ml, 0.30 µg/ml and 1.80 µg/ml), 1.1 (at 0.60 µg /ml), 1.3 (at 0.60 µg/ml) , 2.0 (at 1.20 µg/ml) and 2.2 (at 0.15 µg/ml and 3.00 µg/ml) were observed. In the experiment performed with microsomal activation, comparison of the number of mutant colonies in the controls and in the cultures treated with various concentrations of the test substance revealed no significant deviation, either in these cultures treated with 8-AG or in those screened with 6-TG. The two highest concentrations tested were too toxic and the cultures could not be subjected to the mutant selection procedure. Comparison of the mutant frequency in the control and the treated cultures gave factors of 1.0 (at 0.5 µg/ml, 1.0 µg/ml, 2.0 µg/ml and 4.0 µg/ml) and 1.2 (at 8.0 µg/ml) and 1.5 (at 12.0 µg/ml) in the cultures screened with 8-AG. In the cultures screened with 6-TG factors of 1.0 (at 0.5 µg/ml, 1.0 µg/ml, 2.0 ug/ml, 4.0 µg/ml and 12.0 µg/ml) and 2.6 (at 8.0 µg/ml) were obtained.

All enhancements are regarded as not significant, because the corresponding mutant frequencies are in the range of the historical values obtained from negative controls. Thus, in the investigations performed without and with microsomal activation, a mutant factor greater than 3.0 together with a difference in the treated and untreated dishes of at least 20 clones per 10^6 cells plated was not detectable at any concentration tested, and there was also no indication, based of significantly enhanced mutant frequency values, of a concentration-mutant frequency relation in either experiment. Comparison of the concurrent "positive controls" with EMS (300 nl/ml) and the negative controls in the experiment without microsomal activation gave factors of 287.9 (selection with 8-AG) and 463.8 (selection with 6-TG). In the experiment with metabolic activation, comparison of the mutant frequency values in DMN (1 µl/ml)-treated positive controls and negative controls revealed factors of 33.8 (selection with 8-AG) and 44.8 (selection with 6-TG).

It is concluded, that under the given experimental conditions the substance and its metabolites induced no mutagenic effects in this forward mutation system.

 

In this screening study, the test article, dissolved in culture medium (MEM), was assessed for its potential to induce structural chromosome aberrations in the V79 cell line of the Chinese hamster. The tested concentrations were without S9 mix 1.25, 2.5, 5.0, 10, 20 µg/ml and with S9 mix 1.875, 3.75, 7.5, 15, 30 µg/ml. In the absence and the presence of S9 mix the highest possible test article concentrations were evaluated. Per culture 100 metaphase plates were scored for structural chromosome aberrations. The highest concentration used in a pre-test on toxicity (3400 µg/ml) was chosen with regard to the current OECD-Guideline for in vitro mammalian cytogenetic tests. Dose selection of the cytogenetic experiments was performed considering the toxicity data of the pre-test. In the main experiment, reduced cell numbers were observed in the absence of S9 mix after continuous treatment with 5.0 µg/ml. In the presence of S9 mix after 4 h treatment no clear toxicity was observed at the highest evaluated concentration. Neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test article.

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test article as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test article did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro. Therefore, the substance is considered to be non-mutagenic in this chromosome aberration assay.

Justification for classification or non-classification

Not classifiable