Reaction products of diazotised Sodium Picramate coupled with Resorcinol, subsequently coupled with diazotised 4'-Amino-4-Nitrodiphenylamin-2-Sulfonic Acid, at the end metallized with Iron (II) sulphate heptahydrate, sodium and ammonium salts

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 08 Mar 2018 to 02 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

AcidBrown360

IUPAC Name:

AcidBrown360

Test material form:

solid: particulate/powder

Method

Target gene:

Thymidine kinase enzyme allele on chromosome 11 of mouse lymphoma L5178Y TK+/- (clone 3.7.2C) cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Cells: L5178Y TK+/- mouse lymphoma cells
- Source of cells: American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518)
- Periodically checked for Mycoplasma contamination: yes
- Methods for maintenance in cell culture: permanent stock solutions of the L5178Y TK +/- cells are stored in liquid nitrogen and subcultures are prepared from the frozen stocks for experimental use; prior to use, cells are cleansed of pre-existing mutants
- Generation time and mutation rates: checked at testing facility

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver tissue fraction

Test concentrations with justification for top dose:

- Concentrations:
I experiment
(−S9, 3 h treatment): 830, 692, 576, 480 and 400 µg/mL as test item as received;
(+S9, 3 h treatment): 865, 721, 601, 501, 417 and 348 µg/mL as test item as received.
II experiment
(−S9, 3 h treatment): 346, 288, 240, 200,167, 139 and 116 µg/mLas test item as received;
(+S9, 3 h treatment): 498, 415, 346, 288, 240 and 200 µg/mLas test item as received.

- Justification: 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. Since high levels of cytotoxicity were obtained at all concentrations tested, both in the absence and presence of S9 metabolism, a second mutation assay was performed using lower concentrations.

The test doses were determined in terms of organic content and reported both as organic content and as test item as received. Here are reported as test item as received.

Vehicle / solvent:

- Solvent: RPMI minimal medium
- Justification for choice of solvent/vehicle and for percentage of solvent in the final culture medium:
Solubility of the test item was evaluated in a preliminary trial using DMSO, Ethanol, Acetone and Minimal Medium A. These solvents were selected since they are compatible with the survival of the cells and the S9 metabolic activity. In addition, there are many historical control data demonstrating that no mutagenic effects are induced by these solvents.
Based on the results obtained, DMSO, Ethanol, Acetone were not considered to be suitable vehicles. Using Minimal Medium A, an opaque preparation with moderate precipitation was obtained at 34.6 mg/mL (as test item as received), after mixing and sonication for 5 minutes at 37 °C. An opaque preparation with slight precipitation was noted at 17.3 mg/mL; while at 8.65 mg/mL (as test item as received) an opaque preparation without precipitate was obtained. Suspension at 17.3 mg/mL (organic content), when added to culture medium in the ratio of 1:10, gave an opaque mixture without precipitate. Formulation at 8.65 mg/mL (organic content) was considered suitable for serial dilutions.

Controls

Negative solvent / vehicle controls:

yes

Remarks:

RPMI minimal medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

PREPARATION OF TEST ITEM
Suspensions/solutions of the test item were prepared immediately before use in Minimal Medium A on a weight/volume basis, without correction for the displacement due to the volume occupied by the test item. Suspensions/solutions as organic content were prepared by separate formulations which correspond to 34.6, 17.3, 8.65, 8.30 and 7.47mg/mL, as test item as received. All test item suspensions/solutions were used within 1 hour and 25 minutes from the initial formulation.

PREPARATION OF S9 MIX
One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study (production and quality control certificates indicated suitability) and had the following characteristics:
- Species: rat
- Strain: Sprague-Dawley
- Tissue: liver
- Inducing agents: phenobarbital – 5,6-benzoflavone
- Producer: MOLTOX, Molecular Toxicology, Inc.
- Batch number: 3878
The mixture of S9 tissue fraction and cofactors (S9 mix) was prepared as follows (for each 10 mL):
S9 tissue fraction 0.408 mL
NADP (30 mM) 0.204 mL
G-6-P (590 mM) 0.204 mL
KCl (150 mM) 0.204 mL
Complete medium (5%) 8.98 mL

CULTURE MEDIA
- Minimal medium A
RPMI 1640 (1X) 516.1mL
L-glutamine (200 mM) 5.4mL
Sodium pyruvate (100 mM) 6.0mL
Non-essential amino acids (100X) 5.4mL
Streptomycin sulphate 50000 IU/mL +
Penicillin G 50000 IU/mL 1.1mL
F 68 Pluronic 6.0mL
- Minimal medium B
RPMI 1640 (1X) 522.1mL
L-glutamine (200 mM) 5.4mL
Sodium pyruvate (100 mM) 6.0mL
Non-essential amino acids (100X) 5.4mL
Streptomycin sulphate 50000 IU/mL +
Penicillin G 50000 IU/mL 1.1mL
- Complete medium (5%)
Minimal medium A 950mL
Horse serum (heat-inactivated) 50mL
- Complete medium (10%)
Minimal medium A 900mL
Horse serum (heat-inactivated) 100mL
- Complete medium A (20%)
Minimal medium A 800mL
Horse serum (heat-inactivated) 200mL
- Complete medium B (20%)
Minimal medium B 800mL
Horse serum (heat-inactivated) 200mL

PREPARATION OF CELL CULTURES
A cell suspension (1×106 cells/mL) in complete medium was prepared. A common pool was used for each experiment to prepare the test cultures in appropriately labelled conical screw-cap tissue culture tubes.
The treatment media were prepared as follows:
- Without S9 metabolism - 3 hour treatment time
Cell suspension (1×106 cells/mL in complete medium 5%) 5.0mL
Complete medium (5%) 13.0mL
Solvent/vehicle or Test item solution 2.0mL
20.0mL
- Without S9 metabolism - 24 hour treatment time
Cell suspension (1×106 cells/mL in complete medium 5%) 3.0mL
Complete medium (10%) 15.0mL
Solvent/vehicle or Test item solution 2.0mL
20.0mL
- With S9 metabolism - 3 hour treatment time
Cell suspension (1×106 cells/mL in complete medium 5%) 5.0mL
S9 mix 9.8mL
Solvent/vehicle or Test item solution 2.0mL
Complete medium (5%) 3.2mL
20.0mL
- With S9 metabolism - 3 hour treatment time - positive control
Cell suspension (1×106 cells/mL in complete medium 5%) 10.0mL
S9 mix 9.8mL
Control or Test item solution 0.2mL
20.0mL
The cultures were incubated at 37°C. At the end of the incubation period, the treatment medium was removed and the cultures centrifuged and washed twice with Phosphate Buffered Saline (PBS).

TREATMENT OF CELL CULTURES
Two mutation assays were performed including negative (vehicle) and positive controls. 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), in the absence and presence of metabolic activation.
- Since high levels of cytotoxicity were noted at all concentrations tested, both in the absence and presence of S9 metabolic activation, a second experiment was performed, using the same treatment conditions and a lower range of concentrations.
- After washing in PBS, cells were resuspended in fresh complete medium (10%) and cell densities were determined.
- The number of cells was adjusted to give 2 × 10^5 cells/ml.
- The cultures were incubated at 37 °C in a 5 % CO2 atmosphere (100% nominal relative humidity) to allow for expression of the mutant phenotype.

EXPRESSION PERIOD
- During the expression period (two days after treatment), the cell populations were subcultured in order to maintain them in exponential growth.
- The cell densities of each culture were then determined and adjusted to give 2×10^5 cells/ml.

PLATING FOR 5-TRIFLUOROTHYMIDINE RESISTANCE
- The cell suspensions in complete medium (20%; without F 68 pluronic) were supplemented with trifluorothymidine (final concentration 3.0 μg/ml).
- An estimated 2×10^3 cells were plated in each well of four 96-well plates.
- Plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14-15 days.
- Wells containing clones were identified by eye using background illumination and counted.
- In addition, the number of wells containing large colonies as well as the number of those containing small colonies were scored.

PLATING FOR VIABILITY
- In complete medium (20 %), an estimated 1.6 cells/well were plated in each well of two 96-well plates.
- Plates were incubated at 37 °C in a 5 % CO2 atmosphere (100% nominal relative humidity) for 14-15 days.
- Wells containing clones were identified visually using background illumination and then counted.

Rationale for test conditions:

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 cells used, L5178Y TK+/−, are derived from one of the two clones originated from a thymic tumour induced in a DBA/2 mouse by methylcholanthrene. The use of the TK mutation system in L5178Y mouse lymphoma cells has been well characterised and validated and is accepted by most of the regulatory authorities.
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.

Evaluation criteria:

The assay was considered valid if the following criteria were met:
1. The cloning efficiencies at Day 2 in the solvent control cultures fell within the range of 65-120 %.
2. The solvent control suspension growth over 2 days fell within the range of: i) 8-32 (3 hour treatment), or ii) 32-180 (24 hour treatment).
3. The mutant frequencies in the solvent control cultures fell within the range of 50−170 × 10^−6 viable cells.

The assay was also evaluated as to whether the positive control met at least one of the following two acceptance criteria:
1. The positive control induced a clear increase above the spontaneous background (induced mutent frequency) of at least 300×10^−6. At least 40% of the IMF was reflected in the small colony MF.
2. The positive control induced a clear increase in the small colony IMF of at least 150×10^−6.

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 (126×10^-6) at one or more doses.
2. There is a significant dose-related 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).
- Tests were performed for: i) consistency between plates; ii) heterogeneity factors for replicate cultures; iii) overall consistency; iv) updated heterogeneity factors; v) comparison of each treatment with the control; vi) linear trend

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY CYTOTOXICITY TEST
No precipitation of the test item was noted upon addition of the test item to the cultures. At the end of 3 and 24 hour treatment periods, a dose dependent precipitation of the test item was noted after centrifugation of cultures, at all concentrations tested.
In the absence of S9 metabolic activation, using the 3 hour treatment time, no cells survived treatment at the three highest concentrations tested. No relevant toxicity was noted over the remaining concentrations tested. Using the 24 hour treatment time, no cells survived treatment from 433 μg/mL (as test item as received) on wards, mild toxicity was noted at the next lower concentration (RS=58%), while no relevant toxicity was observed over the remaining dose levels tested.
Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no cells survived treatment at the two highest concentrations tested. Test item treatment at 865 μg/mL yielded marked toxicity reducing RS to 12%, while slight toxicity was observed at the next lower concentration of 433 μg/mL (RS=61%). No toxicity was noted over the remaining concentrations tested.

MAIN EXPERIMENTS (I AND II)
No precipitate was noted upon addition of the test item to the cultures. By the end of treatment period, a coloured pellet was noted after centrifugation of cultures, at all concentrations tested.
Solvent and positive control cultures were included in each mutation experiment. The mutant frequencies in the solvent control cultures fell within the normal range (50−170×10−6 viable cells).
The positive control item met both acceptance criteria:
1. The positive control demonstrated an induced mutant frequency (IMF) of at least 300×10−6; at least 40 % of the IMF was reflected in the small colony MF.
2. The positive control had an increase in the small colony MF of at least 150× 10−6 above that seen in the solvent control.
The cloning efficiencies at Day 2 in the negative control cultures fell within the range of 65 -120 %. The control suspension growth over 2 days fell within the range of 8 - 32 for 3 hour treatments.
Results:
Assay No. S9 Suspension Growth Cloning efficiency (%)
- Experiment I:
(-S9), Suspension Growth 10, Cloning efficiency (%) 93
(+S9), Suspension Growth 12, Cloning efficiency (%) 110
- Experiment II:
(-S9), Suspension Growth 15, Cloning efficiency (%) 105
(+S9), Suspension Growth 12, Cloning efficiency (%) 114

The study was accepted as valid.

SURVIVAL AFTER TREATMENT
- Experiment I
(+S9) no cell survived after treatment at 830 and 692 μg/mL (as test item as received), while severe toxicity reducing Relative Total Growth (RTG) below 10% was noted at the remaining concentrations tested.

(-S9) severe toxicity was noted at the three highest dose levels, marked toxicity reducing RTG to 13% was observed at the next lower concentration of 501 μg/mL (as test item as received), while treatment with the test item at 417 and 348 μg/mL (as test item as received) yielded moderate toxicity, reducing RTG to 25% and 34% of the concurrent negative control value, respectively.

- Experiment II
(-S9) the two highest dose levels yielded moderate toxicity reducing RTG to 24% and 38% of the concurrent negative control value. The next lower dose level of 240 μg/mL (as test item as received) yielded mild toxicity reducing RTG to 45%, while slight toxicity was noted at 200 μg/mL (as test item as received) (RTG = 73%). No relevant toxicity was seen over the remaining dose levels tested.

(+S9) marked toxicity was noted at the highest concentration tested (RTG=14%), while the next two lower dose levels of 415 and 346 μg/mL (as test item as received) yielded moderate toxicity reducing RTG to 30 and 37%, respectively. Mild toxicity was noted over the remaining concentrations tested.

At low survival levels, the mutation data are prone to a variety of artefacts (selection effects, sampling error, founder effects). Mechanisms other than direct genotoxicity per se can lead to positive results that are related to cytotoxicity and not genotoxicity (e.g. events associated with apoptosis, endonuclease release from lysosomes, etc.). For this reason it is generally recommended that such data are treated with caution or excluded from consideration. Accordingly, in the first experiment, we have excluded from the statistical analyses, mutation data obtained in the absence of S9 metabolism at all dose levels and data in the presence of S9 metabolism at the three highest dose levels.

MUTATION
In Mutation Assay I, in the absence of S9 metabolic activation, severe toxicity was observed at all concentrations tested, thus the evaluation of mutagenic effects could not be performed.
In the presence of S9 metabolism, statistically significant increases in mutant frequency were observed at the three analysable concentrations from 348 μg/mL to 865 μg/mL (as test item as received). At the latter concentration, where the RTG was reduced to 13 %, the observed increase was higher than the Global Evaluation Factor (GEF).
In Mutation Assay II, in the presence of S9 metabolism, statistically significant increases in mutant frequency were observed following treatment with the test item at almost all concentrations tested, even though all IMF values were lower than the Global Evaluation Factor (GEF). However, the mutation frequency at the highest concentration of 498 μg/mL (as test item as received) was higher than the recommended acceptable spontaneous mutant frequency (150×10−6)
and fell outside the 95% control limits of the distribution of RTC historical negative control.
A statistically significant dose effect relationship was also indicated by the linear trend analysis. In the absence of S9 metabolism, statistically significant increases in mutant frequency were observed at the three highest dose levels. These increases fell outside the historical background range at the two highest concentrations and a linear trend was indicated. In addition, at 346 μg/mL (as test item as received) (RTG=24%) the induced mutant frequency was higher than the GEF, therefore the results obtained were considered clearly positive.
For the negative and positive controls and for doses showing positive results, the small and large colony mutant frequencies were estimated and the proportion of small mutant colonies was calculated. An adequate recovery of small colony mutants was observed following treatment with the positive controls.

pH AND OSMOLARITY
During the Citotoxicity test dose dependent increases over the concurrent negative controls were observed in all treatment series. However, the results obtained were within acceptable values since they did not increase over 100 mOsm/kg with respect to the negative control values (Scott et al., 1991).
In theMutation Assays, the addition of the test item solutions did not have any obvious effect on the osmolality or pH of the treatment medium.

Applicant's summary and conclusion

Conclusions:

The test item induces mutation at the TK locus of L5178Y mouse lymphoma cells in vitro 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 5-trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method according to the OECD Guideline 490 (2016).

A preliminary solubility trial indicated that the maximum practicable concentration of the test item in the final treatment medium was 3400 μg/mL using Minimal Medium A as solvent. On the basis of this result, a cytotoxicity assay was performed, both in the absence and presence of S9 metabolic activation, using 9 dose levels up to 3460 μg/mL.

Based on the results obtained in the preliminary trial, the first mutation assay was performed using the following dose levels:

(−S9, 3 h treatment): 830, 692, 576, 480 and 400 µg/mL;

(+S9, 3 h treatment): 865, 721, 601, 501, 417 and 348 µg/mL.

In the absence of S9 metabolic activation, severe toxicity, reducing Relative Total Growth (RTG) below 10%, was observed at all concentrations tested, thus the evaluation of mutagenic effects could not be performed. In the presence of S9 metabolism, severe toxicity was noted at the three highest concentrations tested, while marked to moderate toxicity was observed over the three remaining dose levels, where dose related increases in mutation frequency were noted. At the highest analysable concentration (501 μg/mL) the RTG was reduced to 13 % and the observed increase was higher than the Global Evaluation Factor (GEF). Based on the results obtained, in order to evaluate a potential mutagenic effect in a sufficient number of concentrations at adequate levels of cytotoxicity, a second mutation assay was performed, both in the absence and presence of S9 metabolism, using the short treatment time and the following lower range of concentrations:

(−S9, 3 h treatment): 346, 288, 240, 200,167, 139 and 116 µg/mL;

(+S9, 3 h treatment): 498, 415, 346, 288, 240 and 200 µg/mL.

Adequate levels of cytotoxicity, covering a range from the maximum to slight or no toxicity, were observed in both treatment series. In the presence of S9 metabolism, the induced mutation frequency was lower than the Global Evaluation Factor (GEF) at all concentrations tested. However, a statistically significant dose effect relationship was observed and mutation frequency at the highest concentration of 498 μg/mL fell out the distribution of the historical negative control data (95% control limits). In the absence of S9 metabolism, a linear trend was indicated and at the highest dose level, the observed increase was higher than the GEF and thus was considered a clear evidence of positive result.

Appropriate negative and positive control treatments were included in each mutation experiment. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is concluded that the test item induces mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.