Chromate(5-), bis[4-(hydroxy-κO)-7-[2-(2-hydroxy-1-naphthalenyl)diazenyl]-3-[2-[2-(hydroxy-κO)- 3-nitro-5-sulfophenyl]diazenyl]-2-naphthalenesulfonato(4-)]-, sodium (1:5)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

in vitro Bacterial Reverse Mutation, OECD 471 (1983), positive

in vitro Mammalian Chromosomal Aberration, OECD 473 (1983) -V79 cells, negative

in vitro Mammalian Cell Gene Mutation Hprt, OECD 476 (1984)-V79 cells, negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

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)

Version / remarks:

1983

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro mammalian Chromosomal Aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

-Experiment I
without S9 mix:
18 h: 3.0; 10.0; 30.0; 100.0; 300.0; 600.0 µg/ml
28 h: 30.0; 100.0; 300.0; 600.0 µg/ml
with S9 mix:
18 h : 10.0; 30.0; 100.0; 300.0; 600.0; 1000.0 µg/ml
28 h: 100.0; 300.0; 600.0; 1000.0 µg/ml
-Experiment II
with and without S9 mix:
18 h: 0.1; 0.3; 1.0; 3.0; 10.0; 30.0 µg/ml
28 h: 1.0; 3.0; 10.0; 30.0 µg/ml
According to the results from the pre-test 6 concentrations (18 h interval) were chosen to be applied in the chromosomal aberration assay. The highest concentration used in the pre-test (1000.0 µg/ml without S9 mix and 5000.0 µg/ml with S9 mix) was limited by a precipitation of the test substance in culture medium during incubation at 37° C and a strong colouring of the test medium. As strong toxic effects were observed after treatment with 600.0 µg/ml (without S9 mix) and 300,0 µg/ml (with S9 mix) and higher, the cytogenetic experiments were performed with the selected concentrations.

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 Mix

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 Mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
-Cell density at seeding: 5 x 10^5 cells per flask were seeded in 15 ml of MEM (minimal essential medium) supplemented with 10 % fetal calf serum.

CELL CULTURES
Large stocks of the V79 cell line were stored in liquid nitrogen in the cell bank allowing the repeated use of the same cell culture batch in experiments. Before freezing, each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments remained similar because of the reproducible characteristics of the cells. Thawed stock cultures were propagated at 37 °C in 80 cm^2 plastic flasks. The cells per flask were seeded in MEM supplemented with 10 % fetal calf serum. The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C in an atmosphere with 4.5 % carbon dioxide (95.5 % air).

PRE-TEST FOR TOXICITY
High density cultures (approx. 200,000 cells/slide) were treated with the test substance to simulate the conditions of the main experiment. Cell number and morphology were examined qualitatively 4 h and 24 h after the start of treatment (with metabolic activation: after washing with Saline G ) .

DURATION
The treatment interval was 4 h with metabolic activation, 18 h and 28 h without metabolic activation. Per concentration duplicate cultures were used.

DETERMINATION OF CYTOTOXICITY
Toxic effects (low number of remaining/surviving cells, low metaphase number, partially combined with poor metaphase quality) were determined qualitatively for all test groups in both experiments. In the absence of S9 mix, in both experiments cultures after treatment with 30.0 µg/ml (18 h and 28 h) as highest concentration were evaluated for cytogenetic damage. In addition, 10.0 µg/ml and 3.0 µg/ml were evaluated at fixation interval 18 h. In the presence of S9 mix, in experiment I cultures treated with 100.0 µg/ml (18 and 28 h) in experiment II 10.0 µg/ml (18 and 28 h) as top concentrations were evaluated. Additionally, 10.0 and 30.0 ng/ml (exp. I) and 1.0 and 3.0 ng/ml (exp. II), respectively, were evaluated at fixation interval 18 h.

SEEDING OF THE CULTURES
Three days old exponentially growing stock cultures more than 50 % confluent were trypsinized at 37 °C for approximately 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration was 0.2 % in Ca-Mg-free salt solution. The Ca-Mg-free salt solution was composed (per litre) with: NaCl 8000 mg, KCl 400 mg, Glucose 1000 mg, NaHC03 350 mg.
Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/1 EDTA. The cells were seeded into Quadriperm dishes which contained microscopic slides (at least 2 chambers per dish and test group). In each chamber 1 x 10^4 - 6 x 10^4 cells were seeded with regard to preparation time. The medium was MEM + 10 % FCS (complete medium).

TREATMENT
-Exposure time 4 hours (with S9 mix):
In both independent experiments, after 48 h (28 h preparation interval) and 55 h (18 h preparation interval) the culture medium was replaced with serum-free medium containing different concentrations of the test article and 50 µl/ml S9 mix. After 4 h the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.
The "Saline G" solution was composed as (per litre)with: NaCl 8000 mg, KCl 400 mg, Glucose 1100 mg, Na2HP04.7H20 290 mg, KH2PO4 150 mg (pH was adjusted to 7.2).
-Exposure time 18 and 28 hours (without S9 mix):
In both independent experiments, after 48 h (28 h preparation interval) and 55 h (18 h preparation interval) the culture medium was replaced with complete medium (10 % FCS) containing different concentrations of the test article without S9 mix. This medium was not changed until preparation of the cells. All cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % CO2 (95.5 % air).

PREPARATION OF THE CULTURES
15,5 and 25.5 h after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 2.5 h later, the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid. Per experiment both slides per group were prepared. After fixation the cells were stained with Giemsa.

ANALYSIS OF METAPHASE CELLS
Evaluation of the cultures was performed using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To escribe a cytotoxic effect the mitotic Index (% cells in mitosis) was determined. In addition, the number of polyploid cells was scored (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Evaluation criteria:

A test substance was classified as mutagenic if it induced reproducibly either a significant concentration-related increase in the number of structural chromosomal aberrations or a significant and reproducible positive response for at least one of the test points.
A test substance producing reproducibly neither a significant concentration-related increase in the number of structural chromosomal aberrations nor a significant and reproducibly positive response at any one of the test points is considered non-mutagenic in this system. This can be confirmed by means of the chi-square test. However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the chi-square test. Evaluation was performed only for cells carrying aberrations exclusive gaps.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
The concentration range of the test substance set up in the respective experiments were limited by a strong precipitation of the test substance in the test medium during incubation at 37° C and a strong colouring of the test medium. In the main experiments microscopically visible precipitation was found with concentrations starting at 10.0 µg/ml (without S9 mix) and with all concentration used in the presence of S9 mix (both experiments).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxic effects shown by a reduction of mitotic indices and cell density could be observed in exp. I as well as in the pre-experiment in the absence of S9 mix after treatment with 600.0 µg/ml (and higher) at both fixation intervals, in the presence of S9 mix after treatment with 1000.0 µg/ml (18 h) and 600.0µg/ml and higher (28 h).
However, in both experiments lower concentrations were evaluated, where no reduction of mitotic Indices could be found up to the highest concentration evaluated. The evaluation of higher concentrations in the respective intervals were not appropriate due to the strong precipitation of the test substance.
- Other observations:
In both experiments, at both fixation intervals in the absence and presence of S9 mix the test substance did not increase the frequency of cells with aberrations to a biologically relevant extend. The aberration rates of the cells after treatment with the test substance (exp.I: 0.0 % - 2.5 %; exp. II: 0.0 % - 3.0 %) were in or near to the range of the solvent control values (exp. I: 0.0 % - 2.5 %; exp. II: 0.0 % - 2.0 %) and in the range of historical control data: 0.0 % - 4.0 %.
In exp. I, in the presence of S9 mix after treatment with 100.0 µg/ml (fixation interval 28 h) and in exp. II, in the absence of S9 mix after treatment with 3.0 µg/ml (fixation interval 18 h) the statistical evaluation (chi-square test) revealed a significant difference between the treatment groups (2.0 % and 3.0 % aberrant cells) versus the corresponding controls (0.0 %aberrant cells). However, taking into account the extremely low value of the corresponding control and our historical control data range, in this case the statistical significance can be regarded as being biologically not relevant. In both experiments, no biologically relevant increase in rate of polyploid metaphases (exp. I: 1.0 % - 4.5 %; exp. II: 1.5 % -4.5 %) as compared to the rates of the controls (exp. I: 1.0 % -2.5 %; exp. II: 1,0 % - 3.0 %) were found after treatment with the test substance.
POSITIVE CONTROLS
In both experiments, EMS (0.6 mg/ml) and CPA (0.93 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosomal aberrations.

Conclusions:

The substance did not induce reproducibly structural chromosomal aberrations in the V79 Chinese hamster cell line.

Executive summary:

The test item was assessed for its potential to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments, according to the OECD 473 (1983) and the method B.10 of the EEC-Directive 92/69 EEC.The chromosomes were prepared 18 h and 28 h after start of treatment with the test item. The treatment interval was 4 h with metabolic activation (S9 Mix), 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations. The test concentrations, 3 concentrations evaluated at 18 h and the highest evaluable concentration at 28 h, were with and without S9 Mix ranging up to 100 and 30 µg/ml (experiment I) and 10 and 30 µg/ml (experiment II), respectively. The concentration range was limited by a strong precipitation of the test substance in the test medium during incubation at concentrations starting at 10.0 µg/ml in the absence of S9 mix and with all concentrations set up in the presence of S9 mix. Toxic effects could not be found up to the highest concentration evaluated. The evaluation of higher concentrations in the respective intervals was not appropriate due to the strong precipitation of the test substance. In both independent experiments, there were no biologically relevant increases in cells with structural aberrations after treatment with the test substance at both fixation intervals either with or without S9 mix. In both experiments, no biologically relevant increase in the frequency of polyploid metaphases was found after treatment with the test substance compared to the frequencies of the controls. Appropriate reference mutagens were used as positive controls and showed distinct increases in cells with structural chromosomal aberrations.

The substance did not induce structural chromosomal aberrations under the test condition. Therefore, the substance is considered to be non-mutagenic in this chromosome aberration test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

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)

Version / remarks:

1984

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302, L 133, p. 61 - 63

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In vitro Mammalian Cell Gene Mutation Tests

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) locus in V79 cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

-Experiment I: without S9 mix 10.0; 30.0; 100.0; 300.0 and 600.0* µg/ml and with S9 mix 10.0; 100.0; 300.0; 600.0* and 1000.0* µg/ml
-Experiment II: without S9 mix 10.0; 30.0; 100.0; 200.0 and 300.0* µg/ml and with S9 mix 10.0; 100.0; 300.0; 450.0 and 600.0* µg/ml
-Experiment III (additional fourth concentration of experiment I): with S9 mix: 450.0 µg/ml
*not evaluable, toxic effects
According to the recommendations in several guidelines, for example EEC 92/69, the highest concentration should be 10 mM but not higher than 5 mg/ml unless limited by solubility or cytotoxicity of the test article. In case of toxicity the cloning efficiency may be reduced to less than 50 % and/or culture growth at subcultivation should be at least 20 % of the corresponding control.
Due to toxic effects in experiment I the second experiment was performed using a lower highest dose. During the course of the main experiments four concentrations were selected to be evaluated at the end of the experiment concerning concentration range and toxicity. Due to strong toxic effects in experiment I with S9 mix only three concentrations could be evaluated at the end of the experiment. Therefore, a third experiment was performed in order to establish a fourth concentration (450.0 µg/ml) at an appropriate toxicity level (recommended for the determination of a dose-response relationship).

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
MEM (minimal essential medium) supplemented with 10 % fetal calf serum. For the selection of mutants the medium was supplemented with 11 µg/ml thioguanine
-Cell density at seeding: 5 x 10^5 cells seeded into each flask

CELL CULTURES
Large stocks of the V79 cell line were stored in liquid nitrogen in the cell bank allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 °C in 80 cm^2 plastic flasks. The cells were seeded into each flask with 15 ml of MEM, supplemented with 10 % fetal calf serum . The cells are subcultured twice weekly. The cell cultures were incubated at 37 °C in a 4.5 % carbon dioxide atmosphere (95.5 % air). For the selection of mutants the medium was supplemented with 11 µ|/ml thioguanine.

PRE-TEST ON TOXICITY
Two pre-tests were performed in order to determine the concentration range for the mutagenicity experiments. Due to technical reasons these pre-tests could only be evaluated qualitatively.

SEEDING
Three days old exponentially growing stock cultures  (more than 50 % confluent) were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration  for  all  subculturing  steps was  0.2  % in  Ca-Mg-free salt solution.The Ca-Mg-free salt solution was composed (per litre): NaCl 8000 mg, KCl 400 mg, Glucose1000 mg, NaHCO3350 mg. Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA. The cell suspension was seeded into plastic culture flasks. Approximately 1.5 x 106 (single culture) and 5 x 102 cells (in duplícate) were seeded in MEM with 10 % FCS (complete medium) for the determination of mutation rate and toxicity, respectively (see experimental scheme).

TREATMENT
After 24 h the medium was replaced with serum-free medium containing the test substance, either without S9 mix or with 50 µl/ml S9 mix. After 4 h this medium  was replaced with complete medium following two washing steps with "saline G", composed (per litre): NaCl 8000 mg , KCl 400 mg, Glucose 1100 mg, Na2HPO4.7H2O 290 mg, KH2PO4150 mg and pH was adjusted to 7.2
Experimental Scheme included two segments: a)procedure for determination of toxicity and b): Procedure for determination of mutation rates. There were different phases:
1) Subculturing of a log-phase culture. The initial spontaneous mutation rate at the beginning of the experiment of 5.4  (experiment I) and 0.7  (experiment II and III), respectively. a) About 500 cells in 5 ml medium/25 cm2-plastic-flask for cloning efficiency; in duplicate per experimental point rates. b) 1.5 X 106cells in 30 ml medium/175 cm2-plastic-flask for the mutagenicity test, 1 flask per experimental point.
2) Treatment of a) and b)
3) Subculturing of b) in 175 cm2-plastic-flasks 1.5 X 106cells in 30 ml medium/175 cm2-plastic-flasks
4) Fixation and staining of colonies in a)-flasks determination of concentration-related cloning efficiency
5) Subculturing of b) in five 80 cm2-plastic-flasks containing selective medium: mutant selection (about 3-5 x 105cells/flask); subculturing of b) in two 25 cm2-flasks for cloning efficiency (about 500 cells/flask).
6) Fixation and staining of colonies in b)
The cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % CO2. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.The stained colonies with more than 50 cells were counted, in doubt the colony size was checked with a preparation microscope.

Evaluation criteria:

A test substance was classified as positive if it induced either a significant concentration-related increase of the mutant frequency or a reproducible and significant positive response for one of the test points.
A test article producing neither a significant concentration-related increase of the mutant frequency nor a significant and reproducible positive response at any of the test points was considered non-mutagenic in this system.
A significant response was considered if the substance induces reproducibly with one of the concentrations a mutation frequency that is three times higher than the spontaneous mutation frequency in the experiment,
The test substance was classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency.
Such evaluation may be considered also in the case that a three-fold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depended on the level of the corresponding negative control data. If there was by chance a low spontaneous mutation rate in the range normally found ( 0 - 4 5 mutants per 10^ cells) or lower a concentration-related increase of the mutations within this range had to be discussed.

Statistics:

Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method was not available.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The acceptability criteria were complied.

In  experiment  I  and  II, after  treatment  with  concentrations higher than 100.0 µg/ml with and without metabolic activation the cloning efficiency of the cells was reduced. The cell density at first subcultivation in experiment I and in experiment  II was  reduced  after  treatment with concentrations higher than 100.0 µg/ml with and without metabolic activation. At concentrations higher than 10.0 µg/ml finely dispersed particles of the test sunstance could be observed in the culture medium. Increased mutant colony numbers were  found  in the presence of metabolic activation at 450.0 µg/ml (highest concentration) in experiment II and III (obtained mutation factors of 15.8 and  9.5 mutants per 106 cells, respectively). These effects were regarded not to be biologically relevant. Both values of mutant colony numbers (34.8 and 20.9 mutants/per 106 cells, respectively) fell within the range of our histrorical controls of 0-45 mutants per 10⁶cells. Taking into account the mutation rates found in the groups treated with the test substance compared to the negative and solvent controls it can be concluded that no relevant increase of gene mutations was observed. The test substance did not induce a reproducible concentration-related increase in mutant colony numbers. The mutant values of the groups treated with the test substance were in the range of the negative controls. In this study in both experiments (with and without S9 mix) the range of the negative controls was from 2.2 up to 15.5 mutants per 10⁶cells; the range of the groups treated with the test substance was from 0.7 up to 34.8 mutants per 10⁶cells. EMS (0.6 mg/ml) and DMBA (3.85 ng/ml) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:

The test substance did not induce gene mutations at the HPRT locus in V79 cells in this mutagenicity assay and under the experimental conditions.

Executive summary:

The test item was tested for in vitro gene mutation in mammalian cells following the OECD 476 (1984). Chinese Hamster V79 cells were exposed to the test item with and without metabolic activation (S9 mix) at dose ranging up to 1000 and 600 µg/ml (experiment I) and 300 and 600 µg/ml (experiment II), respectively. After the expression period, the mutant frequency was determined by 6 -thioguanine screening and the  substance  resulted  not  having  mutagenic  potential.  Due  to  strong  toxic  effects  in  experiment  I with S9 mix, a third experiment was performed with S9 mix up to a concentration of 450 µg/ml. The concentration ranges for the experiments were selected to yield concentration-related toxic effects. The highest concentration produced a low level of survival and the survival at the lowest concentration was approximately in the range of the negative control. Up to the highest investigated concentration no relevant increase in mutant colony numbers was obtained in two independent experiments. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies.

The substance did not induce gene mutations under the experimental conditions. Therefore, the test item is considered to be non-mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial forward 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:

hamster microsomal fraction S9 mix

Test concentrations with justification for top dose:

33.3; 100.0; 333.3; 1000,0; 2500.0 and 5000.0 µg/plate.
According to the results of the pre-experiment the concentrations applied in the main experiments were chosen. The concentration range covered two logarithmic decades. The maximum concentration was 5000.0 µg/plate. The concentration range included two logarithmic decades. In this study six adequately spaced concentrations were tested. Two independent experiments were performed. As the results of the pre-experiment are in accordance with the criteria described above, these data are reported as a part of the main experiment I.

Vehicle / solvent:

water and DMSO

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent untreated and solvent controls were performed

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine

Remarks:

Without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent untreated and solvent controls were performed

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

other: 2-Aminoanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

TEST SYSTEM
The strains were derived from S. typhimurium strain LT2 and due to mutations in the histidine locus were histidine dependent.
-Storage: the strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.
-Pre-cultures: from the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlemneyer flasks containing 20 ml nutrient medium (this contains per litre 8 g Merck Nutrient Broth, 5 g NaCl).
The bacterial culture was incubated in a shaking water bath for 10 hours at 37 °C.

METHOD OF APPLICATION: in agar
-Selective Agar 2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium. Sterilizations were performed at 121° C in an autoclave.
-The overlay agar contains per litre:
6.0 g Merck Agar Agar
6.0 g NaCl
10.5 mg L-histidine x HCl x H2O
12.2 mg biotin
Sterilizations were performed at 121° C in an autoclave.

Evaluation criteria:

The generally accepted conditions for the evaluation of the results are:
-corresponding background growth on both negative control and test plates
-normal range of spontaneous reversion rates.
Range of spontaneous reversion frequencies
-TA 1535: 10-29
-TA 1537: 5-15
-TA 98: 15-57
-TA 100: 80-123.
Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial assays at this time.
A test substance is considered:
-positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced
-no-mutagenic in this system if produces neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points. A significant response is described:
-a test substance is considered as mutagen if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 it is at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test substance regardless whether the highest dose induced the described enhancement factors or not.

Statistics:

No appropriate statistical method was available.

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Up to the highest investigated dose a clear reproducible dose-dependent increase in revertant colony numbers was observed

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the pre-experiments for toxicity, the plates with the test article showed normal background growth up to 5000.0 µg/plate in strain TA 98 and TA 100, respectively.
No toxic effects, occurred in the test groups with and without metabolic  activation  in experiment  I and  II up to the  highest investigated dose in all strains used. The plates incubated with the test article showed normal back-ground growth up to 5000.0 µg/plate with and without S9 mix in all strains used. Up to the highest investigated dose a clear reproducible dose-dependent increase in revertant colony numbers was observed in strain TA 1537 without S9 mix. No substantial  increases  in revertant colony numbers were observed in the strains TA 1535, TA 98 and TA 100 following treatment  at any dose  level, either  in the presence or 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 significance. The weak  increases  of  revertant  colony  numbers  in  the  strain TA 100 (exp. II, without S9 mix at 5000.0 ug/plate) were considered not to be relevant. The obtained effects were not reproduced in the independent experiment. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced revertant colonies.

Remarks on result:

other: at any dose level

Conclusions:

The substance was considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

The test substance was tested for mutagenic effects with an in vitro bacterial reverse mutation assay using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, according to the OECD Guideline 471 (1983) and the method B.14 of the EEC-Directive 92/69 EEC. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration in the range of 33.3 - 5000 μg/plate and the controls were tested in triplicate. No toxic effects occurred in the test groups with and without metabolic activation in experiment I and II up to the highest investigated dose in all strains used. The plates incubated with the test substance showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used. Up to the highest investigated dose a clear reproducible dose-dependent increase in revertant colony numbers was observed in strain TA 1537 without S9 mix. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. Thus, the test substance induced point mutations by frameshifts in the genome of the strain TA 1537 without metabolic activation, under the experimental conditions.

Therefore, the substance is considered to be mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

BACTERIAL REVERSE MUTATION TEST

The test substance was tested for mutagenic effects with an in vitro bacterial reverse mutation assay using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, according to the OECD Guideline 471 (1983) and method B.14 EEC-Directive 92/69 EEC. The assay was performed in two independent experiments, using identical procedures, both with and without liver microsomal activation. Each concentration in the range of 33.3 - 5000 μg/plate and the controls were tested in triplicate. No toxic effects occurred in the test groups with and without metabolic activation in experiment I and II up to the highest investigated dose in all strains used. The test substance induced point mutations by frameshifts in the genome of the strain TA 1537 without metabolic activation, under the experimental conditions.

IN VITRO MAMMALIAN CHROMOSOMAL ABERRATION TEST

The test item was assessed for its potential to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments, according to the OECD 473 (1983) and the method B.10 of the EEC-Directive 92/69 EEC. The treatment interval was 4 h with metabolic activation (S9 Mix), 18 h and 28 h without metabolic activation. In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosomal aberrations. The test concentrations, 3 concentrations evaluated at 18 h and the highest evaluable concentration at 28 h, were with and without S9 Mix ranging up to 100 and 30 µg/ml (experiment I) and 10 and 30 µg/ml (experiment II), respectively. Toxic effects could not be found up to the highest concentration evaluated. The evaluation of higher concentrations in the respective intervals was not appropriate due to the strong precipitation of the test substance. There were no biologically relevant increases in cells with structural aberrations after treatment with the test substance at both fixation intervals either with or without S9 mix. No biologically relevant increase in the frequency of polyploid metaphases was found after treatment with the test substance compared to the frequencies of the controls. The substance did not induce structural chromosomal aberrations under the test condition.

IN VITRO MAMMALIAN CELL GENE MUTATION TEST USING THE HPRT GENE

The test item was tested for in vitro gene mutation in mammalian cells following the OECD 476. Chinese Hamster V79 cells were exposed to the test item with and without metabolic activation (S9 mix) at dose ranging up to 1000 and 600 µg/ml (experiment I) and 300 and 600 µg/ml (experiment II), respectively. After the expression period, the mutant frequency was determined by 6 -thioguanine screening and the  substance  resulted  not  having  mutagenic  potential.  Due  to  strong  toxic  effects  in  experiment  I with S9 mix, a third experiment was performed with S9 mix up to a concentration of 450 µg/ml. The concentration ranges for the experiments were selected to yield concentration-related toxic effects. The highest concentration produced a low level of survival and the survival at the lowest concentration was approximately in the range of the negative control. Up to the highest investigated concentration no relevant increase in mutant colony numbers was obtained. The substance did not induce gene mutations under the experimental conditions.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008) a mutation means a permanent change in the amount or structure of the genetic material in a cell. The term ‘mutation’ applies both to heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known (including specific base pair changes and chromosomal translocations). The term ‘mutagenic’ and ‘mutagen’ will be used for agents giving rise to an increased occurrence of mutations in populations of cells and/or organisms. For the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- Category 1: substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- Category 2: substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

According to the ECHA Guidance R7.a Table R.7.7-5, in the presence of a positive result in Gene Mutation on bacteria test, further in vitro tests should be performed. In fact, the unique positive response observed in the bacterial test could be due to a specific bacterial metabolism of the test substance. Provided the in vitro tests have given negative results, normally, no in vivo tests would be required. As there are negative results in the “in vitro chromosome aberration” and in the“in vitro gene mutation on mammalian cells” tests, no further testing at this level are necessary and no classification is warranted according to the CLP Regulation (EC 1272/2008).