1-[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl]pyridinium acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

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:

supporting study

Study period:

November 01, 2000 to July 20, 2001

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:

not specified

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Chromosome aberration in chinese hamster ovary (CHO) cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, D-64287 Darmstadt and several times re-cloned
- Storage: in liquid nitrogen in the cell bank of RCC Cytotest Cell Research GmbH allowing the repeated use of the same cell culture batch in experiments
- Suitability of cells: Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Minimal Essential Medium; SEROMED; D-12247 Berlin
- Properly maintained: yes

Thawed stock cultures were propagated at 37° C in 80 cm plastic flasks (GREINER, D-72632 Frickenhausen). About 5x10^ cells per flask were seeded into 15 ml of MEM supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Colbe). The cells were subcultured twice weekly. The cell cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % carbon dioxide (95.5 % air).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver microsomal fraction

Test concentrations with justification for top dose:

160 µg/ml (with and without S9 mix) was chosen as top concentration in the main experiment I.
80 µg/ml were chosen as top treatment concentration for the continuous exposure periods 24 hrs and 46 hrs in the absence of S9 mix. In the presence of S9 mix 50 µg/ml were chosen as top treatment concentration with respect to the results obtained in experiment I.
Due to technical reasons the second experiment was completely repeated with top test item concentrations of 60 µg/ml for continuous treatment without S9 mix and 50 µg/ml for 4 hrs treatment with S9 mix. The concentrations were reduced due to microscopical observations 4 hrs after start of treatment.

Untreated negative controls:

yes

Remarks:

culture medium and solvent

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Remarks:

culture medium and solvent

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Details on test system and experimental conditions:

Reasons for the Choice of the Cell Line CHO
The CHO cell line has been used successfully for many years in in vitro experiments. Especially the high proliferation rate (doubling time of clone CHO/D1 in stock cultures: 16 hrs; determined on February 23, 1996) and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for, the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 21 including one extremely small miniature marker chromosome (microchromosome, centromer region and sister chromatids not always visible).
Lacking metabolic activities of cells under in vitro conditions are a disadvantage of tests with cell cultures as many chemicals only develop a mutagenic potential when they are metabolized by the mammalian organism. However, metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures with liver microsome preparations (S9 mix).

Range-finder
A pre-test on cell growth inhibition with 4 hrs and 24 hrs treatment was performed in order to determine the toxicity of the test item. 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 cultures (seeding 83,000 cells/slide, with regard to the culture time 48 hrs) were treated with the test item for simulating the conditions of the main experiment. A qualitative evaluation of cell number and cell morphology was made 4 hrs and 24 hrs after start of treatment. 24 hrs after start of treatment the cells were stained. Using a 400 fold microscopic magnification the cells were counted in 10 coordinate defined fields of the slides (2 slides per treatment group).
The cell number of the treatment groups is given as % cells in relation to the control.

Dose Selection
The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests. 5000 µg/ml of the test subtance were applied as top concentration for treatment of the cultures in the pre-test. The amount of active ingredient in the test item was 85 %. Test item concentrations between 39.1 and 5000 µg/ml (with and without S9-mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test item after 4 hrs treatment was observed at 156.3 µg/ml and above in the absence of S9 mix and with 39.1 µg/ml and above in the presence of S9 mix.
Using reduced cell numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after treatment with 78.1 µg/ml and above in absence of S9 mix and with 156.3 µg/ml and above in the presence of S9 mix. Considering the test item precipitation and the toxicity data of the pre-test, 160 µg/ml (with and without S9 mix) was chosen as top concentration in the main experiment I.
Dose selection of experiment II was also influenced by test item toxicity observed in the pre-test and in experiment I. In the range finding experiment reduced cell numbers were observed after 24 hrs continuous exposure without S9 mix with 156.3 µg/ml and above. In the first experiment reduced cell numbers were observed after 4 hrs treatment without metabolic activation with 40 µg/ml and above. Therefore, 80 µg/ml were chosen as top treatment concentration for the continuous exposure periods 24 hrs and 46 hrs in the absence of S9 mix. In the presence of S9 mix 50 µg/ml were chosen as top treatment concentration with respect to the results obtained in experiment I. Due to technical reasons the second experiment was completely repeated with top test item concentrations of 60 µg/ml for continuous treatment without S9 mix and 50 µg/ml for 4 hrs treatment with S9 mix. The concentrations were reduced due to microscopical observations 4 hrs after start of treatment.
To verify results obtained in experiment II and to reach a higher toxicity level, a confirmatory experiment, named experiment III, was performed.

Experimental Performance

Schedule
Without S9-mix With S9-mix
exp. I exp. II and III exp. I exp. II and III
Exposure period 4 hrs 24 hrs 46 hrs 4 hrs 4 hrs
Recovery 20 hrs - - 20 hrs 42 hrs
Preparation interval 24 hrs 24 hrs 46 hrs 24 hrs 46 hrs


Seeding of the Cultures

Exponentially growing stock cultures more than 50 % confluent were treated with trypsin 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 (Trypsin: Difco Laboratories, Detroit, USA).
The Ca-Mg-free salt solution was composed as follows (per litre):
NaCl 8000 mg
KCI 400 mg
Glucose 1000 mg
NaHCO3 350 mg

Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA (Ethylene diamine tetraacetic acid).
The cells were seeded into Quadriperm dishes (Heraeus, D-63450 Hanau) which contained microscopic slides (at least 2 chambers per dish and test group). In each chamber 6 x 104 - 9 x 104 cells were seeded with regard to preparation time. The medium was MEM with 10 % FCS (complete medium).

Treatment

Exposure period 4 hours
The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 µl/ml S9 mix per ml culture medium were added. Concurrent negative, solvent and positive controls were performed. After 4 hrs 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 follows (per litre):
NaCl 8000 mg
KCI 400 mg
Glucose 1100 mg
Na2HPO4x7H2O 290 mg
KH2PO4 150 mg

pH was adjusted to 7.2

Exposure period 24 and 46 hours
The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item without S9 mix. The 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
21 hrs and 43 hrs, respectively after the start of the treatment colcemid was added (0.2 µg/ml culture medium) to the cultures. 3 hrs later, the cells on the slides were treated in the chambers with hypotonic solution (0.4 % KCI) 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 preparation the cells were stained
with Giemsa (E. Merck, D-64293 Darmstadt).
Additionally, two cultures per test item and solvent control treatment group, not treated with Colcemid, were set up in parallel. These cultures were stained in order to determine microscopically the cell number within 10 defined fields per slide. The toxicity of the test item is given as reduction of % cells as compared to the solvent control.

Analysis of Metaphase Cells
Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 21 ± 2 were included in the analysis. To describe a cytotoxic effect the mitotic index ( % cells in mitosis) was determined. In addition, the number of polyploid cells was determined
(% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Evaluation criteria:

Evaluation of Results
A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are in the range of our historical control data (0.0 - 4.5 % aberrant cells exclusive gaps). and/or
- no significant increase of the number of structural chromosome aberrations is
A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations are not in the range of our historical control data (0.0 - 4.5 % aberrant cells exclusive gaps). and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05).
However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not cleariy met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05)

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Three independent experiments were performed. In experiment I, the exposure period was 4 hrs with and without metabolic activation. In experiment II and III the exposure periods were 4 hrs with S9 mix and 24 hrs and 46 hrs without S9 mix, respectively. The chromosomes were prepared 24 hrs (exp. I, II, and 111) and 46 hrs (exp. II and III) after start of treatment with the test item. In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations. In a range finding pre-test on toxicity cell numbers 24 hrs after start of treatment were scored as indicator for cytotoxicity. In accordance to the current OECD Guideline 473 the top concentration 5000 µg/ml (4250 µg/ml active ingredient) was chosen. Concentrations between 39.1 and 5000 µg/ml were applied. Clear toxic effects were observed after treatment with 78.1 µg/ml and above in the absence of S9 mix and with 156.3 µg/ml and above in the presence of S9 mix. In addition, 24 hrs continuous treatment with 156.3 µg/ml and above in the absence of S9 mix induced toxic effects. In the pre-test precipitation of the test item in culture medium was observed after treatment with 156.3 µg/ml and above in the absence of S9 mix and 39.1 µg/ml and above in the presence of S9 mix. No relevant influence of the test item on the pH value or osmolarity was observed (solvent control 286 mOsm, pH 7.0 versus 308 mOsm and pH 7.0 at 5000 µg/ml).

In the first cytogenetic experiment after 4 hrs treatment with and without metabolic activation distinct toxicity indicated by reduced celt numbers below 50 % of control were observed with 40 µg/ml and above. In experiment II strongly reduced mitotic indices were observed at 60 µg/ml after continuous treatment in the absence of S9 mix. However, it was impossible to evaluate these cultures due to strong test item induced toxic effects (reduced cell numbers and/or low metaphase numbers, partially paralleled by poor metaphase quality). This was one reason to perform a third experiment. In this experiment strongly reduced cell numbers were observed at 60 µg/ml after continuous treatment in the absence of S9 mix (24 hrs and 46 hrs) and after 4 hrs treatment in the presence of S9 mix.

In three independent experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. In the absence and in the presence of S9 mix, the aberration rates of the cells after treatment with the test item (0.0 % - 3.0 %) were near the range of the solvent control values (0.5 % - 2.5 %) and within the range of our historical control data (0.0 % - 4.5 %). In experiment II an apparent dose dependent increase in the number of aberrant cells was observed after 24 hrs continuous treatment in the absence of S9 mix in a range from 10 to 40 µg/ml (1.0 % up to 3.0 % aberrant cells exclusive gaps). In addition, dose dependence concerning the chromosomal aberration rate was observed after 4 hrs treatment in the presence of S9 mix at the prolonged preparation interval. The increase was observed in the range from 12.5 to 50 µg/ml (1.0 % up to 2.0 % aberrant cells exclusive gaps). However, the corresponding solvent control values (without S9 mix: 2.5 %, with S9 mix 1.5 % aberrant cells exclusive gaps) did not indicate a real dose dependence, but in combination with missing toxicity at the evaluated experimental points, we decided to perform a confirmatory experiment. In this confirmatory experiment narrow concentration steps were used to verify the results and to reach higher toxicity levels. In the third experiment, no dose relation was observed. Therefore, the findings of experiment II were regarded as being biologically irrelevant. In the cytogenetic experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.4 % - 4.3 %) as compared to the rates of the solvent controls (0.4 % - 2.5 %). In the experiments, EMS (300 and 1000 µg/ml, respectively) and CPA (1 µg/ml) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Conclusions:

Non-clastogenic

Executive summary:

Method

The test item, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in CHO cells of the Chinese hamster in vitro, according to the OECD guideline 473, in three independent experiments.

Observation

Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. Clear toxic effects indicated by reduced cell numbers were observed in the absence and in the presence of S9 mix in experiment I after 4 hrs treatment with 40 µg/ml. In experiment III, in the absence of S9 mix strongly reduced cell numbers were observed after 24 and 46 hrs continuous exposure with 60 µg/ml. In addition, reduced cell numbers were observed in the presence of S9 mix after 4 h exposure at preparation interval 46 h. In three independent experiments, neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No biologically relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item 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

Under the experimental conditions reported, the test item did not induce structural chromosome aberrations. Therefore the test substance is considered to be non-clastogenic in this chromosome aberration test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

NR deficient strains

Principles of method if other than guideline:

The traditional strains used for OECD 471 will be checked in parallel with the same strains deficient in the nitro-reductasi enzyme (present only in bacteria) as to avoid the NO2 group reduction present in the test item. Reduction of the nitro groups in fact produce in the substance aromatic amines that typically give false positive for the traditional tested strains.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

nitroreductase deficient

Remarks:

TA98NR, TA100 NR

Metabolic activation:

with and without

Metabolic activation system:

reductive (Prival) metabolic activation system

Test concentrations with justification for top dose:

Highest dose tested: 5000 μg/plate unless limited by cytotoxicity or solubility

Species / strain:

other: all strains

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test is ongoing and is expected to be negative

In the Chromosomal abberration in vitro study the test item , dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in CHO cells of the Chinese hamster in vitro, according to the OECD guideline 473, in three independent experiments.

Dose selection of the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. Clear toxic effects indicated by reduced cell numbers were observed in the absence and in the presence of S9 mix in experiment I after 4 hrs treatment with 40 µg/ml. In experiment III, in the absence of S9 mix strongly reduced cell numbers were observed after 24 and 46 hrs continuous exposure with 60 µg/ml. In addition, reduced cell numbers were observed in the presence of S9 mix after 4 h exposure at preparation interval 46 h. In three independent experiments, neither a significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. No biologically relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item 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.

Under the experimental conditions reported, the test item did not induce structural chromosome aberrations.

Therefore the test substance is considered to be non-clastogenic in this chromosome aberration test.

In vivo Genetic Toxicity

In a NMRI mouse bone marrow micronucleus test conducted in accordance with OECD Guideline 474, 5 mice/sex/treatment group were treated intraperitoneally once with the test item (95.6% purity) at doses of 0 and 50 mg/kg bw. The vehicle used was physiological saline. The animals were sacrificed and bone marrow cells were harvested after 16 hours (test item), 24 hours (negative/positive control and test item) and after 48 hours (test item).

After application the animals showed the following signs of toxicity: apathy, roughened fur, staggering gait, spasm, twitching, difficulty in breathing and orange discoloured urine. The test item was tested at an adequate dose based on a preliminary dose range finding test. The positive control induced the appropriate response.

There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time (16, 24 and 48 hours).

Justification for classification or non-classification

GERM CELL MUTAGENICITY

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

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

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

The classification in Category 2 is based on:

— positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:

— somatic cell mutagenicity tests in vivo, in mammals; or

— other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.

Note: Substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.

Classification for heritable effects in human germ cells is made on the basis of well conducted, sufficiently validated tests as:

- In vivo somatic cell mutagenicicty tests such as these indicated in paragraph 3.5.2.3.5:

— mammalian bone marrow chromosome aberration test;

— mouse spot test;

— mammalian erythrocyte micronucleus test.

- In vitro mutagenicity tests such as these indicated in 3.5.2.3.8:

— in vitro mammalian chromosome aberration test;

— in vitro mammalian cell gene mutation test;

— bacterial reverse mutation tests.

Ames test is ongoing and is expected to be negative. An in vitro cytogenicity test is available and the test substance does not show any mutagenic effect.

As per REACH Regulation requirements, a further in vivo study is considered in which the test substance didn’t induce increase the number of polychromatic cells with micronuclei.

Therefore, as reported in the Flow chart R.7.7 -1, no further test are required and it is possible to conclude that the test substance is Not Classified for Mutagenic Toxicity.