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Genetic toxicity in vitro

Description of key information
Acid Red EAY 9656 induces a marginal, not reproducible, and in the preincubation assay not dose-dependent, mutagenic effect in Salmonella typhymurium TA98 in the absence and presence of S9 metabolism. Consequently, no clear conclusion can be drawn from the bacterial mutation assay and the result was considered ambiguous in this assay. As a consequence the test substance was tested additionally in a mammalian mutation assay. Acid Red EAY 9656 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. Hence the test substance is considered to be not mutagenic.
Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
June 2015 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant with GLP and testing guidelines; coherence among data, results and conclusions.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
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
Target gene:
The test item Acid Red EAY 9656 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. HPRTdeficient 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 / 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 3417 and 3453
Test concentrations with justification for top dose:
A preliminary cytotoxicity assay was performed at the following dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1 and 19.5
μg/mL
Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels:
Main Assay I (-S9): 1000, 500, 250, 125, 62.5 and 31.3 μg/mL
Main Assay I (+S9): 5000, 4000, 3200, 2560 and 2050 μg/mL
Main Assay II (-S9): 1000, 769, 592, 455, 350 and 269 μg/mL
Main Assay II (+S9): 2000, 1000, 500, 250 and 125 μg/mL
Vehicle / solvent:
Test item solutions were prepared using EMEM minimal medium.
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. In this test a wide range of dose
levels of the test item was used. cell cultures were treated using the same treatment conditions as the mutation assays, and the survival of the cells
was subsequently determined. 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. On the day before the experiment, sufficient numbers of 75 cm^2 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: At Day 1 a number of cells was replated in order to maintain the treated cell populations. On Day 3, the cell populations were subcultured in order to maintain them in exponential growth. When Day 9 is used as expression time, subculturing was performed also at Day 6.
Determination of mutant frequency: A single expression time was used for each experiment: Day 9 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:
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.
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, in the absence of S9 metabolism, slight to moderate reduction in relative survival was noted starting from 62.5 μg/mL, while severe toxicity (RS = 2%) was observed at the highest dose level of 5000 μg/mL. In the presence of S9 metabolism, slight to mild reduction in relative survival was noted at the four highest dose levels, while no relevant toxicity was observed at 2050 μg/mL.
In Main Assay II, in the absence of S9 metabolism, dose related toxicity was noted starting from 455 μg/mL and a severe reduction of relative survival
(RS = 9%) was observed at the highest dose level. In the presence of S9 metabolism, no toxicity was noted at any concentration tested. By the end of
treatment, no precipitation was observed at any dose level in the absence of S9 metabolism. In its presence, test item treatment mixture at 2000 μg/mL appeared as gelatinous suspension; while no precipitation was noted over the remaining concentrations tested.
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 dose level and replicate culture were not significant factors in explaining the observed variation in the data, in the absence and presence of S9 metabolism, in Main Assay I and II.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that Acid Red EAY 9656 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 Acid Red EAY 9656 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 EMEM minimal medium. A preliminary cytotoxicity assay was performed. The test item was assayed, in the absence and presence of S9 metabolism, at a maximum dose level of 5000 μg/mL (the upper limit indicated in the Study Protocol) and at a wide range of lower dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1 and 19.5 μg/mL. No precipitation was noted in the absence of S9 metabolism. By the end of treatment, in the presence of S9 metabolism, test item treatment mixtures at the two highest dose levels appeared as gelatinous suspensions, while no precipitation was noted at the remaining concentrations tested. In the absence of S9 metabolism, reduction in percentage of survival was noted starting from 78.1 μg/mL, severe toxicity was observed at the three highest dose levels. In the presence of S9 metabolism, survival was reduced to 29% of the concurrent negative control value at the highest dose level, while no relevant toxicity was noted over the remaining concentrations tested. Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels described in the following table:

Main Assay I (-S9): 1000, 500, 250, 125, 62.5 and 31.3 μg/mL

Main Assay I (+S9): 5000, 4000, 3200, 2560 and 2050 μg/mL

Main Assay II (-S9): 1000, 769, 592, 455, 350 and 269 μg/mL

Main Assay II (+S9): 2000, 1000, 500, 250 and 125 μg/mL

Selection of dose levels used in Main Assay I was performed in order to investigate the test item at adequate level of cytotoxicity. The dose range used in Main Assay II was modified taking into account precipitation and toxicity observed by the end of treatment in the previous Main Assay. 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 Acid Red EAY 9656 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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

 

 

The test item Acid Red EAY 9656 was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. An additional experiment was performed in the presence of metabolic activation, using uniduced liver S9 fraction from the Chinese hamster with the pre-incubation method (Prival modification). The test item was used as a solution in dimethylsulfoxide (DMSO).

Toxicity test: The test item Acid Red EAY 9656 was assayed in the toxicity test at a maximum concentration of 5000 μg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. No toxicity was observed with any tester strains in the absence or presence of S9 metabolism.

Main Assays : On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625, 313 μg/plate. No precipitation of the test item was observed at the end of the incubation period at any concentration. No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism. The test item induced increases in the number of revertant colonies in the absence and presence of S9 metabolism with TA98 tester strain, reaching a two-fold increase at the highest concentration tested.

As this increase was only small and only valid for the vehicle but not for the negative control, a confirmatory assay was performed in which TA98 tester strain was treated in the absence and presence of S9 metabolism at the dose levels of 5000, 3330, 2220, 1480, 988 and 658 μg/plate. In this assay, the mutation frequency (MF) with metabolic activation was 1.87, thus negative, without metabolic activation the mutation frequency was 2.029 for the vehicle control and 1.8 for the negative control, thus questionable.

As the outcome of the assay was unclear, an additional experiment (Main Assay III) was performed in which all tester strains were treated in the presence of S9 metabolism by using the preincubation method and the Prival metabolizing system at the dose levels of 5000, 3330, 2220, 1480, 988 and 658 μg/plate. In this assay, increases in revertant numbers were observed with TA98 tester strain which reached twice the vehicle control value (highest MF 2.4 to vehicle control and 1.9 to negative control); however, these increases were not dose-related. Hence, this assay did not meet the criterion for a positive outcome with respect to the dose-dependency of mutation frequencies.

In conclusion, the test item Acid Red EAY 9656 induces a marginal, not reproducible, and in the preincubation assay not dose-dependent, mutagenic effect in Salmonella typhymurium TA98 in the absence and presence of S9 metabolism. Consequently, no clear conclusion can be drawn from the bacterial mutation assay and the result was considered ambiguous in this assay. As a consequence the test substance was tested additionally in a mammalian mutation assay.

The test item Acid Red EAY 9656 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 EMEM minimal medium. A preliminary cytotoxicity assay was performed. The test item was assayed, in the absence and presence of S9 metabolism, at a maximum dose level of 5000 μg/mL (the upper limit indicated in the Study Protocol) and at a wide range of lower dose levels: 2500, 1250, 625, 313, 156, 78.1, 39.1 and 19.5 μg/mL. No precipitation was noted in the absence of S9 metabolism. By the end of treatment, in the presence of S9 metabolism, test item treatment mixtures at the two highest dose levels appeared as gelatinous suspensions, while no precipitation was noted at the remaining concentrations tested. In the absence of S9 metabolism, reduction in percentage of survival was noted starting from 78.1 μg/mL, severe toxicity was observed at the three highest dose levels. In the presence of S9 metabolism, survival was reduced to 29% of the concurrent negative control value at the highest dose level, while no relevant toxicity was noted over the remaining concentrations tested. Two independent assays for mutation to 6-thioguanine resistance were performed using dose levels described in the following table:

Main Assay I (-S9): 1000, 500, 250, 125, 62.5 and 31.3 μg/mL

Main Assay I (+S9): 5000, 4000, 3200, 2560 and 2050 μg/mL

Main Assay II (-S9): 1000, 769, 592, 455, 350 and 269 μg/mL

Main Assay II (+S9): 2000, 1000, 500, 250 and 125 μg/mL

Selection of dose levels used in Main Assay I was performed in order to investigate the test item at adequate level of cytotoxicity. The dose range used in Main Assay II was modified taking into account precipitation and toxicity observed by the end of treatment in the previous Main Assay. 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 Acid Red EAY 9656 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.

Justification for classification or non-classification

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