N-alkylester N-substituted heterocyclic diazo aryl amine

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 03 March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

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 (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

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 and 3263

Test concentrations with justification for top dose:

A preliminary cytotoxicity assay was performed at the following dose levels: 625, 313, 156, 78.1, 39.1, 19.5, 9.77, 4.88 and 2.44 µg/mL.
Two independent assays for mutation to 6-thioguanine resistance were performed using the following concentrations:
Main Assay I (+S9): 625, 313, 156, 78.1, and 39.1 µg/mL
Main Assay I (-S9): 156, 78.1, 39.1, 19.5, and 9.77 µg/mL
Main Assay II (+S9): 156, 78.0, 39.0, 19.5 and 9.75 µg/mL
Main Assay II (-S9): 39.0, 19.5, 9.75, 4.88 and 2.44 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:

A preliminary cytotoxicity test was undertaken in order to select appropriate dose levels for the mutation assays.
Treatments were performed both in the absence and presence of S9 metabolism; 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. 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.
Determination of mutant frequency: A single expression time was used for each experiment: Day 6 in Main Assay I and Day 8 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 105 cells were plated in each of five 100 mm tissue culture petri dishes containing medium supplemented with 6-thioguanine (at 7.5 µg/mL). 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:

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

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Survival after treatment: No relevant toxicity was observed at any concentration tested, in any experiment in the absence or presence of S9 metabolis.
Mutation results: No relevant increases over the spontaneous mutation frequency were observed in any experiment, at any treatment level either in the absence or presence of S9 metabolic activation. 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, in Main Assay I in the absence of S9 metabolism.

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item 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. Based on solubility features, the test item was assayed at a maximum dose level of 625 µg/mL and at a wide range of lower dose levels: 313, 156, 78.1, 39.1, 19.5, 9.77, 4.88 and 2.44 µg/mL. No relevant toxicity was observed at any concentration tested, in the absence or presence of S9 metabolism. A coloured film, adhering to the flask surface, was noted starting from 39.1 µg/mL, in the absence of S9 metabolism and at the three highest dose levels in the presence of S9 metabolism.

Two independent assays for mutation to 6-thioguanine resistance were performed using the following concentrations:

Main Assay I (+S9): 625, 313, 156, 78.1, and 39.1 µg/mL

Main Assay I (-S9): 156, 78.1, 39.1, 19.5, and 9.77 µg/mL

Main Assay II (+S9): 156, 78.0, 39.0, 19.5 and 9.75 µg/mL

Main Assay II (-S9): 39.0, 19.5, 9.75, 4.88 and 2.44 µg/mL

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.