N-[2-[(2-chloro-4,6-dinitrophenyl)azo]-5-(diallylamino)-4-methoxyphenyl]acetamide

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 December 2014 to 04 May 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

methyl 2-({4-[2-(2-bromo-4,6-dinitrophenyl)diazen-1-yl]-5-acetamido-2-methoxyphenyl}(2-methoxy-2-oxoethyl)amino)acetate

EC Number:

808-279-8

Cas Number:

88938-51-6

Molecular formula:

C21H21BrN6O10

IUPAC Name:

methyl 2-({4-[2-(2-bromo-4,6-dinitrophenyl)diazen-1-yl]-5-acetamido-2-methoxyphenyl}(2-methoxy-2-oxoethyl)amino)acetate

Test material form:

solid: particulate/powder

Details on test material:

see below

Method

Target gene:

The test item was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment.
6-thioguanine can be metabolised by the enzyme hypoxanthine-guaninphosphoribosyl-transferase (HPRT) into nucleotides, which are used in nucleic acid synthesis resulting in the death of HPRT-competent cells. HPRT-deficient cells, which are presumed to arise through mutations in the HPRT gene, cannot metabolise 6-thioguanine and thus survive and grow in its presence.

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: EMEM medium supplemented with 10% Foetal Calf Serum (EMEM complete)
- Properly maintained: yes; permanent stock of V79 cells are stored in liquid nitrogen and subcoltures are prepared from the frozen stocks for experimental use.
- Periodically checked for Mycoplasma contamination: yes
- The karyotype, 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: Phenobarbital – 5,6-Benzoflavone Producer: MOLTOX, Molecular Toxicology, Inc. Batch Numbers. 3332, 3263 and 3417

Test concentrations with justification for top dose:

A preliminary cytotoxicity assay was performed at the following dose levels: 1250, 625, 313, 156, 78.1, 39.1, 19.5, 9.77 and 4.88 µg/mL.
A second toxicity test was performed in the absence of S9 metabolism using the following lower and closer concentrations: 10.0, 7.14, 5.10, 3.64 and 2.60 µg/mL.
Since no adequate toxicity for dose selection was obtained, a third toxicity test was performed in the absence of S9 metabolism, using the following concentrations: 40.0, 20.0, 10.0, 5.00 µg/mL.
Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels:
Main Assay I (+S9): 600, 400, 267, 178, 119 and 79.0 µg/mL
Main Assay I (-S9): 40.0, 20.0, 10.0, 5.00, 2.50, 1.25 and 0.625 µg/mL
Main Assay II (+S9): 400, 308, 237, 182, 140 and 108 µg/mL
Main Assay II (+S9): 20.0, 15.4, 11.8, 9.10, 7.00 and 5.39 µg/mL

Vehicle / solvent:

Test item solutions were prepared using dimethylsulfoxide (DMSO).

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Preliminary cytotoxicity tests were undertaken in order to select appropriate dose levels for the mutation assays: a single experiment was performed in the presence of S9 metabolism, while three tests were performed in the absence of S9 metabolic activation.A single culture was used at each test point and positive controls were not included.
Two Mutation Assays were performed including negative 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. On the day before the experiment, sufficient numbers of 75 cm2 flasks were inoculated with 2 million freshly trypsinised V79 cells from a common pool. The cells were allowed to attach overnight prior to treatment. Following treatment, the cultures were incubated at 37°C for three hours. At the end of the incubation period, the treatment medium was removed and the cell monolayers were washed with PBS. Fresh complete medium was added to the flasks which were then returned to the incubator at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.
Determination of survival: The following day, the cultures were trypsinised and an aliquot was diluted and plated to estimate the viability of the cells.
Subculturing: On Day 3, the cell populations were subcultured in order to maintain them in exponential growth. When Day 8 was used as expression time, subculturing was performed on Day 4 and Day 6.
Determination of mutant frequency: A single expression time was used for each experiment: Day 8 in Main Assay I and Day 6 in Main Assay II. At the expression time, each culture was trypsinised, resuspended in complete medium and counted by microscopy. After dilution, an estimated 1 x 10^5 cells were plated in each of five 100 mm tissue culture petri dishes containing medium supplemented with 6-thioguanine.
These plates were subsequently stained with Giemsa solutions and scored for the presence of mutants. After dilution, an estimated 200 cells were plated in each of three 60 mm tissue culture petri dishes. These plates were used to estimate Plating Efficiency (P.E.).

Evaluation criteria:

EVALUATION CRITERIA
For a test item to be considered mutagenic in this assay, it is required that:
- There is a five-fold (or more) increase in mutation frequency compared with the solvent controls, over two consecutive doses of the test item. If only the highest practicable dose level (or the highest dose level not to cause unacceptable toxicity) gives such an increase, then a single treatment-level will suffice.
- There must be evidence for a dose-relation (i.e. statistically significant effect in the ANOVA analysis).

Statistics:

The results of these experiments were subjected to an Analysis of Variance in which the effect of replicate culture and dose level in explaining the observed variation was examined. For each experiment, the individual mutation frequency values at each test point were transformed to induce homogeneous variance and normal distribution. The appropriate transformation was estimated using the procedure of Snee and Irr (1981), and was found to be y = (x + a)b where a = 0 and b = 0.275. A two way analysis of variance was performed (without interaction) fitting to two factors:
- Replicate culture: to identify differences between the replicate cultures treated.
- Dose level: to identify dose-related increases (or decreases) in response, after allowing for the effects of replicate cultures and expression time.
The analysis was performed separately with the sets of data obtained in the absence and presence of S9 metabolism.

Results and discussion

Test results

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:

Survival after treatment:
In Main Assay I, following treatment in the absence of S9 metabolism, a severe toxic effect was observed at the highest dose level (40.0 µg/mL) reducing survival to 2% of the concurrent negative control value. At the next lower concentration of 20.0 µg/mL, survival was reduced to 13%. Mild toxicity was observed at 10.0 µg/mL, while no relevant toxicity was noted at the remaining concentrations tested. In the presence of S9 metabolic activation, severe toxicity (relative survival < 10%) was observed at the two highest dose levels (600 and 400 µg/mL), while test item treatment at 267 µg/mL yielded 41% relative survival. At lower dose levels, no relevant toxicity was noted. At the highest dose level tested, no cells were recovered on Day 6 both in the absence and presence of S9 metabolic activation. Since at low survival levels, mutation results are prone to a number of artefacts (selection effects, sampling error, founder effects) and mechanisms other than direct genotoxicity per se can lead to positive results (e.g. events associated with apoptosis, endonuclease release from lysosomes, etc.), the dose level of 400 µg/mL was excluded from analysis.
In Main Assay II, in the absence of S9 metabolism, survival was reduced to 15% at the highest dose level of 20.0 µg/mL; moderate toxicity (approximately 30% of RS) was noted at the two next lower concentrations; mild toxicity (RS=58%) was observed at 9.10 µg/mL, while no relevant toxicity was observed at the remaining dose levels. It should be noted that on Day 6 a lower number of cells, compared to the negative control value, was recovered at the highest dose level. In the presence of S9 metabolism, test item treatment at 400 µg/mL yielded 31% relative survival; mild toxicity (RS = 63%) was noted at the next lower dose level, while no relevant toxicity was observed over the remaining concentrations.
Mutation results:
In the presence of S9 metabolism, no increase over the spontaneous mutation frequency was observed in any experiment, at any treatment level. In the absence of S9 metabolic activation, no increase over the spontaneous mutation frequency was observed in Main assay I (Day 8), while a dose related increase in mutation frequency, which reached five-fold the concurrent negative control value at 15.4 µg/mL, was observed in Main Assay II (Day 6). Analysis of variance indicated that replicate culture was not a significant factor in explaining the observed variation in the data, in the absence and presence of S9 metabolism, in Main Assay I and II. Dose level was a significant factor (p < 0.05%) in explaining the observed variation in the data of Main Assay II in the absence of S9 metabolism.

Applicant's summary and conclusion

Conclusions:

The test substance does not induce mutation in Chinese hamster V79 cells after in vitro treatment, in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Executive summary:

The test item was examined for mutagenic activity by assaying for the induction of 6-thioguanine resistant mutants in Chinese hamster V79 cells after in vitro treatment. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. Test item solutions were prepared using dimethylsulfoxide (DMSO). A preliminary cytotoxicity assay was performed, in the absence and presence of S9 metabolic activation. Based on solubility features, the test item was assayed at a maximum dose level of 1250 µg/mL and at a wide range of lower dose levels: 625, 313, 156, 78.1, 39.1, 19.5, 9.77 and 4.88 µg/mL. In the absence of S9 metabolism, no cells survived at almost all the concentrations tested; survival was reduced to 50% of the concurrent negative control value at the lowest dose level (4.88 µg/mL). In the presence of S9 metabolism, severe toxicity was noted at the two highest concentrations, while survival was reduced to 44% of the concurrent negative control value at 313 µg/mL. No relevant toxicity was noted over the remaining concentrations tested. By the end of treatment time, precipitation of test item and/or a coloured film, adhering to the flask surface, was observed at all concentrations tested in the absence of S9 metabolism and at the three highest dose levels in its presence. In order to select the appropriate range of concentrations for the mutation assay, an additional toxicity test was performed in the absence of S9 metabolism using the following lower and closer concentrations: 10.0, 7.14, 5.10, 3.64 and 2.60 µg/mL. Since no adequate toxicity for dose selection was obtained, a third toxicity test was performed in the absence of S9 metabolism, using the following concentrations: 40.0, 20.0, 10.0, 5.00 µg/mL. Severe toxicity was noted at the highest concentration; survival was reduced to 12% and 37% of the concurrent negative control value at 20.0 and 10.0 µg/mL, respectively; while no relevant toxicity was observed at 5.00 µg/mL. Two independent assays for mutation to 6-thioguanine resistance were performed using the following dose levels:

Main Assay I (+S9): 600, 400, 267, 178, 119 and 79.0 µg/mL

Main Assay I (-S9): 40.0, 20.0, 10.0, 5.00, 2.50, 1.25 and 0.625 µg/mL

Main Assay II (+S9): 400, 308, 237, 182, 140 and 108 µg/mL

Main Assay II (+S9): 20.0, 15.4, 11.8, 9.10, 7.00 and 5.39 µg/mL

In the second experiment the concentration range was slightly modified on the basis of the toxicity results obtained in Main Assay I. No reproducible five-fold increases in mutant numbers or mutant frequency were observed following treatment with the test item at any dose level, in the absence or presence of S9 metabolism. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is concluded that the substance does not induce gene mutation in Chinese hamster V79 cells after in vitro treatment in the absence or presence of S9 metabolic activation, under the reported experimental conditions.