Trisodium 5-[[4-chloro-6-(ethylphenylamino)-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[(4-methyl-2-sulphonatophenyl)azo]naphthalene-2,7-disulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- The test substance was assayed for mutagenicity by the bacterial Reverse Mutation Test. The test was performed according to the method OECD No.: 471. The test substance was not mutagenic in this Ames test.

- The test substance was assayed for mutagenicity by in human lymphocytes. The test was performed according to the method OECD No.: 487. The test substance was not mutagenic in this test.

- The test substance was assayed for mutagenicity by induction of 5 trifluorothymidine resistant mutants in mouse lymphoma L5178Y cells. The test was performed according to the method OECD No.:490. The test substance was not mutagenic in this test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1.4.-6.5.1994

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:

not specified

GLP compliance:

no

Type of assay:

bacterial gene mutation assay

Target gene:

gene for synthesis histidine

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

other: histidine dependent strain

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: histidine dependent strain

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: histidine dependent strain

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: histidine dependent strain

Metabolic activation:

with and without

Metabolic activation system:

supernatant of rat liver and mixture of cofactors

Test concentrations with justification for top dose:

10-5000 µg per plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminofluorene

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS:
Two series of experiments were performed with each strain - without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule, its using is comperable with using of statiistical method (3,4). After this rule the result is positive, when reproducible dose-efftect and/or doubling of ratio Rt/Rc is reached.

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the above-described experimental design, the test substance was non mutagenic for all Salmonella typhimurium strain in experiments without as well as with metabolic activation.

Executive summary:

The test substance was assayed for mutagenicity by the bacterial Reverse Mutation Test.

Four indicator Salmonell typhimurium strains TA 98, TA 97, TA 100 and TA 1535 strai were used. The test substance was dissolved in water and assayed in doses of 10 -5000 µg which were applied to plate. Two series of experiments were performed with each strain without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance was nonmutagenic for all the used bacterial strains with as well as without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21.01.-12.04.2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

yes

Remarks:

see 'Principles of method if other than guideline'

Principles of method if other than guideline:

Due to a technical oversight, the following two deviations occurred during the study:
– For the short term treatment in the presence of S9, check of osmolality of treatment media was not performed concurrently with treatment of cell cultures, but as a separate experimental phase,
– For the continuous treatment in the absence of S9, a total of 494 cells and not 500 cells were examined for the determination of CBPI of one culture (no. 52).
In addition, for the short term treatment both in the absence and presence of S9, due to the prolonged washing procedures, harvesting of cells was performed 33 hours and not 32 hours after beginning of treatment.
For one blood sample, concentration of heparin was slightly higher than the one indicated in the Study Protocol (616 IU/mL of blood instead of approximately 556 IU/mL).
All deviations were not considered to have affected the integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: human whole blood provided by Biopredic International (France).

For lymphocytes:
- Sex, age and number of blood donors: One male, 32 years old; one female 27 years old.
- Whether whole blood or separated lymphocytes were used: whole blood was used.
- Whether blood from different donors were pooled or not: yes
- Mitogen used for lymphocytes: phytohaemagglutinin (10 mL to 500 mL medium)

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: RPMI 1640 1x (Dutch modification) 500 mL, Foetal Calf Serum (heat-inactivated) 100 mL, L-Glutamine (200 mM) 6.25 mL, Antibiotic solution 1.25 mL

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : Liver S9 from male Sprague Dawley rat, induced by phenobarbital and 5,6-benzoflavone.
- Method of preparation of S9 mix : 1.0 mL S9 tissue fraction, 0.4 mL NADP (100 mM), 0.5 mL G-6-P (100 mM), 0.2 mL MgCl2 (100 mM), 5.0 mL phosphate buffer (pH 7.4, 200 mM) and 2.9 mL distilled water.
- Concentration or volume of S9 mix and S9 in the final culture medium: 1 mL S9 mix added to 0.5 mL test item solution (or solvent/vehicle) + 3.5 mL culture medium. Thus 20% (v/v) S9 mix.
- Quality controls of S9: Alkoxyresorufin-O-dealkylase activities, promutagen activation.

Test concentrations with justification for top dose:

52.0, 78.0, 117, 176, 263, 395, 593, 889, 1330 and 2000 µg/mL

Vehicle / solvent:

Solvent: culture medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

cyclophosphamide

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Three treatment series were performed including negative and positive controls. Two replicate cultures were prepared at each test point

METHOD OF TREATMENT/ EXPOSURE:
- Lymphocyte cultures were treated for 48 hours after they were initiated. Before treatment, cultures were centrifuged at 1000 rpm for 10 minutes and the culture medium was decanted and replaced with treatment medium.
- For the short treatment series, the treatment media were added to the tubes and the cultures were incubated for 3 hours at 37°C. At the end of treatment time, the cell cultures were centrifuged and washed twice with Phosphate Buffered Saline Solution. Fresh medium was added and the cultures were incubated for a further 28 hours (Recovery Period) before harvesting. At the same time, Cytochalasin-B was added to achieve a final concentration of 6 μg/mL.
- For the continuous treatment series, 3 hours after beginning of treatment, Cytochalasin-B was also added and the cultures were incubated for a further 28 hours before harvesting.
- Using the short treatment time, since tests with and without metabolic activation were done concurrently, positive control cultures were treated only with Cyclophosphamide at the dose levels of 20.0 and 15.0 μg/mL.
- Using the long treatment time, in the absence of S9 metabolism, the positive control cultures were treated with Colchicine at the dose levels of 80.0 and 40.0 ng/mL.
- The lymphocyte cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant was removed. The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension. A few drops of the cell suspension obtained in this way were dropped onto clean, wet, grease free glass slides. Three slides were prepared for each test point and each was labelled with the identity of the culture. The slides were allowed to air dry and kept at room temperature prior to staining with a solution of Acridine Orange in PBS.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- The cytokinesis-block proliferation index CBPI was calculated and used to measure the cytotoxic effect. Five hundred cells per cell culture were analysed and when negligible cytotoxicity was observed, scoring was interrupted. The highest dose level for genotoxicity assessment was selected on the basis of the cytotoxicity as calculated by the CBPI. The highest dose level for genotoxicity assessment was selected as a dose which produces a substantial cytotoxicity compared with the solvent control. In the absence of cytotoxicity, the highest treatment level is selected as the highest dose level for scoring. Two lower dose levels were also selected for the scoring of micronuclei. For the three selected doses, for the solvent and the positive control Cyclophosphamide, 1000 binucleated cells per cell culture were scored to assess the frequency of micronucleated cells.

SCORING METHOD FOR MICRONUCLEI:
1. The micronucleus diameter was less than 1/3 of the nucleus diameter
2. The micronucleus diameter was greater than 1/16 of the nucleus diameter
3. No overlapping with the nucleus was observed
4. The aspect was the same as the chromatin

Evaluation criteria:

Acceptance criteria:
The assay is considered valid if the following criteria are met:
– The incidence of micronucleated cells of the negative control is within the distribution range of our historical control values.
– Concurrent positive controls induce responses that are compatible with those generated in our historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
– Adequate cell proliferation is observed in solvent control cultures.
– The appropriate number of doses and cells is analysed.

Criteria for outcome:
In this assay, the test substance is considered as clearly positive if the following criteria are met:
– Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
– The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values (95% control limits).
– There is a significant dose effect relationship.

The test substance is considered clearly negative if the following criteria are met:
– None of the dose levels shows a statistically significant increase in the incidence of micronucleated cells.
– There is no concentration related increase when evaluated with the Cochran-Armitage trend test.
– All the results are inside the distribution of the historical control data (95% control limits).

Statistics:

For the statistical analysis, a modified χ2 test was used to compare the number of cells with micronuclei in control and treated cultures.
Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES
The test substance was found to be soluble in culture medium at 20.0 mg/mL (in terms of test substance as supplied). On the basis of this result, the maximum dose level of 2000 μg/mL was employed for the cytogenetic assay. Following treatment with the test substance, no precipitate visible by eye was noticed at the beginning or by the end of treatment, in the absence or presence of S9 metabolism. Following treatment with the test substance, no remarkable variation of pH or osmolality was observed at any dose level, in the absence or presence of S9 metabolism. Following treatment with the test substance, no remarkable cytotoxicity was seen over the whole dose range in any treatment series. On the basis of this result, the dose levels of 2000, 1330 and 889 μg/mL were selected for the scoring of micronuclei.

STUDY RESULTS:
Concurrent vehicle negative and positive control data Results show that the incidence of micronucleated cells of the negative controls was within the 95% control limits of the distribution range of our historical control values. Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system and the responses were compatible with those generated in our historical control database. The study was accepted as valid.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements: Following treatment with the test substance, no statistically significant increase over the concurrent negative control was observed in any treatment series. All incidences were within the normal distribution of historical control data (95% confidence limits) with the exception of the result obtained at the highest dose level using the short term treatment in the absence of S9 metabolism. This value slightly exceeded the upper confidence limit for this treatment series, but fell within the 95% control limits of the other two series. In addition, no concentration related increase was seen, hence this result was considered without any biological relevance. On the basis of the above mentioned results and in accordance with the criteria for outcome of the study, the test substance was not considered to induce micronuclei in human lymphocytes after in vitro treatment.

Conclusions:

On the basis of the results, it is concluded that the test substance does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Executive summary:

The test substance was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation (according to OECD TG 487 and GLP principles). Three treatment series were performed. A short term treatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of 33 hours, corresponding to approximately two cell cycle lengths, was used. A long term (continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours. Solutions of the test item were prepared in culture medium. Dose levels for treatment were selected on the basis of the solubility test. The maximum dose level of 2000 μg/mL was chosen in agreement with the Study Protocol. Lower dose levels of 1330, 889, 593, 395, 263, 176, 117 and 78.0 μg/mL were used for the three hour treatment in the absence and presence of S9 metabolism. For the continuous treatment, the additional dose level of 52.0 μg/mL was included. 

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrent solvent control value was observed at any dose level, in any treatment series. All incidences were within the distribution of historical negative control values (95% control limits) with the exception of the result obtained at the highest dose level using the short term treatment in the absence of S9 metabolism. This value slightly exceeded the upper confidence limit for this treatment series, but fell within the 95% control limits of the other two series and no concentration related increase was seen. Hence, this result was considered without any biological relevance.

It is concluded that the test substance does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25.03-23.05.2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type of cells and source: The cells used, L5178Y TK+/-, are derived from one of the two clones derived 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 many regulatory authorities. The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory

MEDIA USED
- Permanent stocks of the L5178Y TK+/- cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use. Cultures of the cells are grown in RPMI 1640 minimal medium supplemented with 10% horse serum heat-inactivated at 56 °C for 20 minutes before use (Complete medium 10%). The incubations are at 37 °C in a 5% carbondioxide atmosphere (100% nominal relative humidity).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9 : The rat S9 liver tissue fraction will be obtained from an appropriate supplier. Induction of drug metabolising enzyme-levels is routinely
performed using Phenobarbital and 5,6-Benzoflavone (Mixed Induction).
- Method of preparation of S9 mix : The mixture of S9 tissue fraction and cofactors (S9 mix) will be prepared in the following proportions: S9 tissue fraction 0.408 mL, NADP (0.03 M) 0.204 mL, G-6-P (0.59 M) 0.204 mL, KCl (150 mM) 0.204 mL, Complete medium (5%) 8.98 mL

Test concentrations with justification for top dose:

Assay no 1: 255, 511, 1020, 1280, 1600, 2000 µg/mL (without metabolic activity)
Assay no 1: 128, 255, 511, 1024, 1280, 1600 µg/mL (with metabolic activity)
Assay no 2: 255, 511, 1020, 1280, 1600 and 2000 µg/,L (with and without metabolic activity)

Vehicle / solvent:

- solvent used: RMPI 1640 Minimal

Untreated negative controls:

yes

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:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate were prepared at each test point with the exception of the positive controls which were prepared in a single culture.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 2×10^5 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration: 3 hour (first experiment) and 24 hour (second experiment)

OTHER:
Expression period:
During the expression period (two days after treatment), the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determined and adjusted to give 2×10^5 cells/mL.

Plating for 5-trifluorothymidine resistance:
After dilution, the cell suspensions in complete medium B (20%) were supplemented with trifluorothymidine (final concentration 3.0 μg/mL) and 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 days and 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:
After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of two 96-well plates. These plates were incubated at 37°C in a 5% CO2 atmosphere (100% nominal relative humidity) for 14 days and wells containing clones were identified as above and counted.

Measurements of cytotoxicity:
Cloning efficiencies in test and control cultures were compared to give the relative cloning efficiency (RCE). The survival calculation takes into account post-treatment relative cloning efficiencies and reduction in post-treatment cell count.
In order to aid toxicity data interpretation, the relative total growth (RTG), expressed as a percentage of the concurrent negative control, was also calculated. This is a product of the relative suspension growth (RSG) and the Day 2 relative cloning efficiency (RCE), expressed as a percentage of the concurrent negative control.

Evaluation criteria:

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

Every 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 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 induced a clear increase in the small colony IMF of at least
150×10^6.


Criteria for outcome assay:
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-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).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No remarkable variation was observed at any dose level, in the absence or presence of S9 metabolism
- Data on osmolality: No remarkable variation was observed at any dose level, in the absence or presence of S9 metabolism
- Precipitation and time of the determination: Following treatment with the test item, no precipitate visible by eye was noticed at the beginning or by the end of treatment, in the absence or presence of S9 metabolism.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Results show that the incidence of micronucleated cells of the negative controls was within the 95% control limits of the distribution range of our historical control values. Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system and the responses were compatible with those generated in our historical control database. The study was accepted as valid.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
Following treatment with the test item, no remarkable cytotoxicity was seen over the whole dose range in any treatment series. On the basis of this result, the dose levels of 2000, 1330 and 889 μg/mL were selected for the scoring of micronuclei.

- Genotoxicity results:
Following treatment with the test item, no statistically significant increase over the concurrent negative control was observed in any treatment series. All incidences were within the normal distribution of historical control data (95% confidence limits) with the exception of the result obtained at the highest dose level using the short term treatment in the absence of S9 metabolism. This value slightly exceeded the upper confidence limit for this treatment series, but fell within the 95% control limits of the other two series. In addition, no concentration related increase was seen, hence this result was considered without any biological relevance. On the basis of the above mentioned results and in accordance with the criteria for outcome of the study, the test item was not considered to induce micronuclei in human lymphocytes after in vitro treatment.

Conclusions:

It is concluded that the test substance does not induce 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 substance 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 OECD TG 490 and GLP principles). Based on solubility data, the maximum practicable concentration of the test item in the final treatment medium was 2000 μg/mL using RPMI 1640 Minimal as solvent. On the basis of this result, a preliminary cytotoxicity assay was performed both in the absence and presence of S9 metabolic activation, using the maximum dose level of 2000 μg/mL and a wide range of lower dose levels: 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 μg/mL. Furthermore two independent assays for mutation at the TK locus were performed with the following concentrations: 255, 511, 1020, 1280, 1600, 2000 µg/mL (without metabolic activity, assay 1), 128, 255, 511, 1024, 1280, 1600 µg/mL (with metabolic activity, assay 1) and 255, 511, 1020, 1280, 1600 and 2000 µg/mL (with and without metabolic activity, assay 2).

In the absence of S9 metabolic activation, using the 3 hour treatment time, moderate to mild reduction of Relative Survival (RS) was observed at the two highest concentrations of 2000 and 1000 μg/mL, while no relevant toxicity was noted at the remaining dose levels. Using the 24 hour treatment time, moderate toxicity was observed at the three highest dose levels, while mild reduction of RS was seen at the remaining concentrations tested. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no cells survived treatment at 2000 µg/mL, while moderate to mild reduction of RS was seen at the next two lower dose levels of 1000 and 500 µg/mL. No relevant toxicity was observed over the remaining dose levels. Regarding to the independent assay for mutation at the TK locus, the presence of S9 metabolism the concentration of 1600 µg/mL did not elicit adequate level of cytotoxicity and no precipitation or turbidity was seen. Since this concentration was lower than the maximum dose level indicated in the relevant guideline, a repeated experiment in the presence of S9 metabolism was performed, using 2000 µg/mL as top concentration. The treatment series using a longer treatment time (24 hours), in the absence of S9 metabolic activation, was carried out at the same time. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. 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. No relevant increases in mutant frequencies were observed following treatment with the test item, in any treatment series.

It is concluded that the test substance does not induce 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

OECD 471:

The test substance was assayed for mutagenicity by the bacterial Reverse Mutation Test.

Four indicator Salmonella typhimurium strains TA 98, TA 97, TA 100 and TA 1535 strain were used. The test substance was dissolved in water and assayed in doses of 10 -5000 µg which were applied to plate. Two series of experiments were performed with each strain without metabolic activation and with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance was non-mutagenic for all the used bacterial strains with as well as without metabolic activation.

 

OECD 487:

The test substance was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation (according to OECD TG 487 and GLP principles). Three treatment series were performed. A short term treatment, where the cells were treated for 3 hours, was performed in the absence and presence of S9 metabolism. The harvest time of 33 hours, corresponding to approximately two cell cycle lengths, was used. A long term (continuous) treatment was also performed only in the absence of S9 metabolism, until harvest at 31 hours. Solutions of the test item were prepared in culture medium. Dose levels for treatment were selected on the basis of the solubility test. The maximum dose level of 2000 μg/mL was chosen in agreement with the Study Protocol. Lower dose levels of 1330, 889, 593, 395, 263, 176, 117 and 78.0 μg/mL were used for the three hour treatment in the absence and presence of S9 metabolism. For the continuous treatment, the additional dose level of 52.0 μg/mL was included. 

One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells. Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicine, indicating the correct functioning of the test system. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrent solvent control value was observed at any dose level, in any treatment series. All incidences were within the distribution of historical negative control values (95% control limits) with the exception of the result obtained at the highest dose level using the short term treatment in the absence of S9 metabolism. This value slightly exceeded the upper confidence limit for this treatment series, but fell within the 95% control limits of the other two series and no concentration related increase was seen. Hence, this result was considered without any biological relevance.

It is concluded that the test substance does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

 

OECD 490:

The test substance 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 OECD TG 490 and GLP principles). Based on solubility data, the maximum practicable concentration of the test item in the final treatment medium was 2000 μg/mL using RPMI 1640 Minimal as solvent. On the basis of this result, a preliminary cytotoxicity assay was performed both in the absence and presence of S9 metabolic activation, using the maximum dose level of 2000 μg/mL and a wide range of lower dose levels: 1000, 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 μg/mL. Furthermore two independent assays for mutation at the TK locus were performed with the following concentrations: 255, 511, 1020, 1280, 1600, 2000 µg/mL (without metabolic activity, assay 1), 128, 255, 511, 1024, 1280, 1600 µg/mL (with metabolic activity, assay 1) and 255, 511, 1020, 1280, 1600 and 2000 µg/mL (with and without metabolic activity, assay 2).

In the absence of S9 metabolic activation, using the 3 hour treatment time, moderate to mild reduction of Relative Survival (RS) was observed at the two highest concentrations of 2000 and 1000 μg/mL, while no relevant toxicity was noted at the remaining dose levels. Using the 24 hour treatment time, moderate toxicity was observed at the three highest dose levels, while mild reduction of RS was seen at the remaining concentrations tested. Following treatment in the presence of S9 metabolic activation, using the short treatment time (3 hours), no cells survived treatment at 2000 µg/mL, while moderate to mild reduction of RS was seen at the next two lower dose levels of 1000 and 500 µg/mL. No relevant toxicity was observed over the remaining dose levels. Regarding to the independent assay for mutation at the TK locus, the presence of S9 metabolism the concentration of 1600 µg/mL did not elicit adequate level of cytotoxicity and no precipitation or turbidity was seen. Since this concentration was lower than the maximum dose level indicated in the relevant guideline, a repeated experiment in the presence of S9 metabolism was performed, using 2000 µg/mL as top concentration. The treatment series using a longer treatment time (24 hours), in the absence of S9 metabolic activation, was carried out at the same time. Negative and positive control treatments were included in each mutation experiment in the absence and presence of S9 metabolism. 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. No relevant increases in mutant frequencies were observed following treatment with the test item, in any treatment series.

It is concluded that the test substance does not induce 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.

Justification for classification or non-classification

Based on the available data, the test substance is not classified for mutagenicity.