[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl]trimethylammonium chloride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

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

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

other: experimental result on similar substance

Adequacy of study:

key study

Study period:

2015-12-16 to 2016-05-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

The mutation assay method used in this study is based on the identification of L5178Y colonies which have become resistant to a toxic thymidine analogue trifluorothymidine (TFT). This analogue can be metabolised by the enzyme thymidine kinase (TK) into nucleosides, which are used in nucleic acid synthesis resulting in the death of TK-competent cells. TK-deficient cells, which are presumed to arise through mutations in the TK gene, cannot metabolise trifluorothymidine and thus survive and grow in its presence. In the L5178Y mouse lymphoma cells, the gene which codes for the TK enzyme is located on chromosome 11. Cells which are heterozygous at the TK locus (TK+/-) may undergo a single step forward mutation to the TK-/- genotype in which little or no TK activity remains. The mouse lymphoma assay often produces a bimodal size distribution of TFT resistant colonies designated as small or large. It has been evaluated that point mutations and deletions within the active allele (intragenic event) produce large colonies. Small colonies result in part from lesions that affect not only the active TK allele but also a flanking gene whose expression modulates the growth rate of cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium supplemented with 10% Foetal Calf Serum (RPMI complete)
- Properly maintained: yes; permanent stock of mouse lymphoma L5178Y cells are stored in liquid nitrogen and subcultures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The generation time, plating efficiency and mutation rates (spontaneous and induced) have been checked in this laboratory.
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 tissue fraction: Species: Rat, Strain: Sprague Dawley, Tissue: Liver, Inducing Agents: P henobarbital – 5,6-Benzoflavone, Producer: MOLTOX, Molecular Toxicology, Inc. Batch Number 3512

Test concentrations with justification for top dose:

A preliminary cytotoxicity assay was performed at the following dose levels: 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81 and 3.91 μg/mL. Based on the results obtained in the preliminary cytotoxicity assay, two independent assays for mutation at the TK locus were performed using the following dose levels:
Main Assay I (-S9; treatment time 3 hours): 60.2, 43.0, 30.7, 22.0, 15.7 and 11.2 μg/mL
Main Assay I (+S9; treatment time 3 hours): 100, 50.0, 25.0, 12.5, 6.25 and 3.13 μg/mL
Main Assay II (-S9; treatment time 24 hours): 35.0, 25.0, 17.9, 12.8, 9.11 and 6.51 μg/mL

Vehicle / solvent:

Test item solutions were prepared using dimethylsulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

Details on test system and experimental conditions:

A preliminary cytotoxicity test was performed in order to select appropriate dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined. Treatments were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9 metabolic activation. A single culture was used at each test point. The mutation assays were performed including vehicle and positive controls, in the absence and presence of S9 metabolising system. Duplicate cultures were prepared at each test point, with the exception of the positive controls which were prepared in a single culture. In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained without metabolic activation, the second experiment in the absence of S9 metabolism was performed, using a longer treatment time (24 hours). After washing in Phosphate Buffered Saline (PBS), cells were resuspended in fresh complete medium (10%) and incubated to allow expression of the mutant phenotype. At the end of the expression period cells were plated for the evaluation of 5-trifluorothymidine resistance and for viability.

Rationale for test conditions:

For a test item to be considered mutagenic in this assay, it is required that:
1. The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126x10^-6) at one or more doses.
2. There is a significant dose-relationship as indicated by the linear trend analysis.
Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.

Statistics:

Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990).

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Survival after treatment:
In the first experiment, in the absence of S9 metabolic activation, no cell survived after treatment at the highest dose level, moderate toxicity reducing relative total growth (RTG) to 14% of the concurrent negative control was noted at 43.0 μg/mL, slight to mild toxicity was observed between 15.7 and 30.7 μg/mL, while no relevant toxicity was noted at the lowest concentration tested. In the presence of S9 metabolism, treatment with the test item at 100 μg/mL yielded moderate toxicity reducing RTG to 24% of the concurrent negative control value, slight toxicity was noted at the next lower concentration, while no relevant toxicity was observed over the remaining concentrations tested. In the second experiment, in the absence of S9 metabolic activation using a long treatment time, the highest dose level selected (35 μg/mL) yielded marked toxicity reducing RTG to 8% of the concurrent negative control value. The next two lower dose levels of 25.0 and 17.9 μg/mL yielded moderate toxicity reducing RTG to 15 and 26%, respectively. Dose-related toxicity was seen over the remaining dose levels tested.

Mutation results:
In Main Assay I, statistically significant increases in mutant frequency were observed at the lowest and at two intermediate concentrations, in the absence of S9 metabolism, and at the highest dose level in its presence. A linear trend was indicated, both in the absence and presence of S9 metabolism. However, the observed increases were lower than the Global Evaluation Factor both in the absence and presence of S9 metabolism, thus they were considered of no biological relevance. In Main Assay II, no increases in mutant frequency were observed at any concentration tested.

Conclusions:

Under the experimental conditions reported, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay using mouse lymphoma L5178Y cells.

Executive summary:

In a mammalian cell gene mutation assay conducted according to OECD 490, mouse lymphoma L5178Y cells cultured in vitro were exposed to the test item (87.9% purity) in dimethylsulfoxide at concentrations of 11.2, 15.7, 22.0, 30.7, 43.0 and 60.3 μg/mL (main assay I, without S9 mix) and at 3.13, 6.25, 12.5, 25.0, 50.0 and 100 μg/mL (main assay I, with S9 mix) and in main assay II, without S9 mix at concentrations of 6.51, 9.11, 12.8, 17.9, 25.0 and 35.0 μg/mL. The test item was tested in the main assay I up to 60.3 μg/mL (without metabolic activation) and up to 100 μg/mL (with metabolic activation) and in main assay II up to 35.0 μg/mL (without metabolic activation) based on data obtained from the preliminary cytotoxicity test. The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 490

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

other: experimental result on similar substance

Adequacy of study:

key study

Study period:

1992-03-17 to 1993-01-11

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian germ cell cytogenetic assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: F. Winkelmann, Borchen, Germany
- Age at study initiation: 8 - 12 weeks of age
- Weight at study initiation: 28 - 43 g
- Housing: females: in groups of a maximum of three mice; males: individually in Macrolon type I cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1.5
- Humidity (%): 40 -70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in physiological saline solution, stirred with a magnetic mixer during administration and injected intraperitoneally.

Duration of treatment / exposure:

test item dose groups: 16, 24 and 48 hours,
negative/positive control: 24 hours

Frequency of treatment:

once

Post exposure period:

no

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

three test item related experimental dose groups: 16, 24 and 48 hours

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control used: cyclophosphamide
- Route of administration: intraperitoneal
- Dosis: 20 mg/kg bw

Tissues and cell types examined:

bone marrow derived erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The selection of the dose was based on a pilot test, in which groups of five animals, including both males and females, were i.p. administered 10 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 75 mg/kg and 100 mg/kg bw. For the results please box "Additional information on results".

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
see Table 1 in box "Any other information on materials and methods incl. tables".

DETAILS OF SLIDE PREPARATION:
Bone marrow was flushed into a tube containing fetal bovine serum and centrifuged (5 min, 1000 rpm)
Air dried smears were automatically stained with an Ames HemaTek Slide Stainer and then destained with methanol, rinsed with deionized water and dried. After drying, the slides were covered with xylene and a cover glass.

METHOD OF ANALYSIS:
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. Moreover, the ratio of polychromatic to normochromatic erythrocytes was determined (number of normochromatic erythrocytes per 1000 polychromatic ones). Additionally, the number of normochromatic erythrocytes showing micronuclei was also established.

Evaluation criteria:

A test was considered positive if at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control. A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory’s experience, was within the range of negative controls. A test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group.

Statistics:

The test item group(s) with the highest mean and the positive control were checked by Wilcoxon’s non parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi²-test. A variation was considered statistically significant, if the error probability was below 5% and the treatment group figure was higher than that of the negative control. In addition, standard deviations (1s ranges) were calculated for all the means.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
The selection of the dose was based on a pilot test, in which groups of five animals, including both males and females, were i.p. administered 10 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 75 mg/kg and 100 mg/kg bw. The following symptoms were recorded for up to 48 hours, starting at 10 mg/kg bw: apathy, roughened fur, red discoloration of hairless parts of skin, staggering gait, spasm, leaping coloured urine. In addition, 3 of 5 animals died in the 75 mg/kg group and all animals died in the 100 mg/kg bw group. Based on the results, 50 mg/kg bw was considered as 1 MTD for the main test.

RESULTS OF DEFINITIVE STUDY
- Clinical signs: After application the animals showed the following signs of toxicity: apathy, roughened fur, staggering gait, spasm, twitching, difficulty in breathing and orange discoloured urine. No symptoms were recorded for the control groups.
- Mortality: One of forty treated animals died during the test period due to acute toxicity. No animals died in the control group.
- Induction of micronuclei (for Micronucleus assay): no clastogenic effect, see Table 2
- Ratio of PCE/NCE (for Micronucleus assay): slightly changed, see Table 2
- Statistical evaluation: see Table 2

Table 2: Summary of results of micronucleus test with the test item
experimental groups		Number of evaluated poly-chromatic erythrocytes (PCE)		Number of normo-chromatic erythrocytes per 1000 PCE		micronucleated cells per 1000
						normo-chromatic erythrocytes		poly-chromatic erythrocytes
Negative control		10000		811 +/- 208		1.2 +/- 1.6		1.5 +/- 1.1
Test item_16 hours		10000		1321* +/- 319		1.3 +/- 1.0		1.6 +/- 1.0
Test item_24 hours		10000		1088 +/- 549		1.3 +/- 1.6		1.5 +/- 1.0
Test item_48 hours		10000		775 +/- 199		0.4 +/- 0.7		1.3 +/- 1.2
Positive control		10000		557 +/- 231		0.4 +/- 1.3		12.6* +/- 6.9

Concentration: test item: 50 mg/kg bw; positive control: 20 mg/kg bw

* p< 0.01 (tested by non-parametric Wilcoxon ranking test)

Conclusions:

Non-mutagenic with respect to clastogenicity and aneugenicity in the mammalian erythrocyte micronucleus test.

Executive summary:

Method

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.

Results

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).

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Ames test is on going and expetced to be negative.

In the Gene mutation in vitro study the substance was tested according to OECD 490, mouse lymphoma L5178Y cells cultured in vitro were exposed to the test item in dimethylsulfoxide at concentrations of 11.2, 15.7, 22.0, 30.7, 43.0 and 60.3 μg/mL (main assay I, without S9 mix) and at 3.13, 6.25, 12.5, 25.0, 50.0 and 100 μg/mL (main assay I, with S9 mix) and in main assay II, without S9 mix at concentrations of 6.51, 9.11, 12.8, 17.9, 25.0 and 35.0 μg/mL. The test item was tested in the main assay I up to 60.3 μg/mL (without metabolic activation) and up to 100 μg/mL (with metabolic activation) and in main assay II up to 35.0 μg/mL (without metabolic activation) based on data obtained from the preliminary cytotoxicity test.

The positive controls did induce the appropriate response.There was no evidence of induced mutant colonies over background.

In vivoGenetic 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).