Registration Dossier

Administrative data

Endpoint:
genetic toxicity in vivo, other
Remarks:
single cell gell / Comet assay
Type of information:
experimental study planned
Study period:
not yet defined
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS


NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out : Acid Brown 360

CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION [please address all points below]:
- Available GLP studies : assessment on genotoxic potential of test substance was initially based on th following tests:
MUTATION IN L5178Y TK+/− MOUSE LYMPHOMA CELLS (OECD 490)
IN VITRO MICRONUCLEUS TEST IN HUMAN LYMPHOCYTES (OECD 487)
- Available non-GLP studies : Not Available
- Historical human data : Not Available
- (Q)SAR : Not Available
- In vitro methods : assessment on genotoxic potential of test substance was initially based on th following tests:
MUTATION IN L5178Y TK+/− MOUSE LYMPHOMA CELLS (OECD 490)
IN VITRO MICRONUCLEUS TEST IN HUMAN LYMPHOCYTES (OECD 487)
- Weight of evidence: available results from in vitro studies (GLP) were both taken into account, but no definitive conclusion could be drawn
- Grouping and read-across: no read across data available

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- According to the Annex VIII, Column 2 of the REACH Regulation, appropriate in vivo studies shall be considered in case of positive result in any of the genotoxicity studies in Annex VII or VIII. Specific adaptation possibilities apply in case of in vivo studies already available.
Based on the expected positive result in the Ames test, on the positive result in the mouse lymphoma assay and on the lack of results from in vivo studies, an appropriate in vivo genotoxicity study is proposed.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed: single cell gel / Comet assay.
Such method allows to measure DNA strand breaks. DNA strand breaks may result from direct interactions with DNA, alkali labile sites or as a consequence of incomplete excision repair. Therefore, the alkaline comet assay recognises primary DNA damage that would lead to gene mutations and/or chromosome aberrations, but will also detect DNA damage that may be effectively repaired or lead to cell death. The comet assay can be applied to almost every tissue of an animal from which single cell or nuclei suspensions can be made, including specific site of contact tissues.
As reported in literature, from the analysis of 91 chemicals with published data from both tests, the comet assay appears to yield similar results to the TGR assay in liver and gastrointestinal tract; hence the comet assay in an acceptable choice when the intention is to confirm in vitro gene mutation activity in terms of genotoxicity in general
Given that the substance showed positive results in the MLA and is highly likely to be mutagenic in the Ames test, it should be necessary to consider performing an in vivo mammalian alkaline comet assay in rats following recommendations stated in ECHA's 2015 REACH progress report.

Cross-referenceopen allclose all
Reason / purpose:
other: reference to other assay considered in the justification for the testing proposal
Reference
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
Qualifier:
according to
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
Target gene:
Thymidine kinase enzyme allele on chromosome 11 of mouse lymphoma L5178Y TK+/- (clone 3.7.2C) cells
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.
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
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.
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.

Reason / purpose:
other: reference to other assay considered in the justification for the testing proposal
Reference
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
from 28 Jan. 2019 to 08 Mar. 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016
Deviations:
no
Principles of method if other than guideline:
Since the test item gave positive results in a mouse lymphoma mutation assay without any remarkable difference with and without S9 metabolic activation, a direct genotoxic mechanism was suspected. This study was intended as a further investigation to evaluate cytogenetic effects and treatments were performed only in the absence of S9 metabolism.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human whole body
Details on mammalian cell type (if applicable):
-Source: Biopredic International (France)
- Batches used:
(i)
Sex Female
Age 33 years old
Donor information healthy, no smoker without any recent exposure to drugs or radiation
Anticoagulant Sodium heparin, 556 IU/mL of whole blood
Collection date 24 January 2019
Code Number 2019/05
(ii)
Sex Female
Age 30 years old
Donor information healthy, no smoker without any recent exposure to drugs or radiation
Anticoagulant Sodium heparin, 556 IU/mL of whole blood
Collection date 24 January 2019
Code Number 2019/06

- Culture medium composition:
RPMI 1640 1x (Dutch modification) 500mL
Foetal Calf Serum 100mL
L-Glutamine (200mM) 6.25mL
Antibiotic solution 1.25mL
The foetal calf serum was heat-inactivated at 56 °C for 20 minutes before use. For the initiation of the cultures, medium with the addition of phytohaemagglutin (PHA) was used in the following proportion: 10 mL of PHA was added to 500 mL of medium.
Metabolic activation:
without
Metabolic activation system:
Positive control: S9
Test concentrations with justification for top dose:
Dose levels were selected on the basis of the cytotoxicity results obtained in MLA Study No. A3170.
- short term treatment series:
1250, 826, 556, 371, 247, 165, 110, 73.2 and 48.8 µg/mL (expressed as organic conten)
1730, 1140, 769, 513, 342, 228, 152, 101 and 67.5 µg/mL (expressed as test item as received)
- continuous treatment:
1250, 826, 556, 371, 247, 165, 110, 73.2, 48.8 and 32.5 µg/mL (expressed as organic conten)
1730, 1140, 769, 513, 342, 228, 152, 101, 67.5 and 45.0 µg/mL (expressed as test item as received)
Vehicle / solvent:
- Test item: Minimal Medium A (RTC batch nos.: 008/222 and 008/225).
- Benzo(a)pyrene (B(a)P): DMSO (batch no.: H012S, obtained from Honeywell)

Preparation of the test item
Suspensions/solutions of the test item were prepared immediately before use in culture medium on a weight/volume basis, without correction for the displacement due to the volume occupied by the test item. Concentrations were expressed in terms of organic content. Suspensions/solutions at 12.5, 8.26 and 5.56 mg/mL (as organic content), were prepared by separate formulations (corresponding to 17.3, 11.4 and 7.69 mg/mL, as test item as received). All test item suspensions/solutions were used within 45 minutes from the initial formulation.
Treatment with the positive control Colchicine was performed under safe light conditions.
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
colchicine
mitomycin C
Details on test system and experimental conditions:
- Preparation of the test cultures and treatment
Two treatment series were performed including negative and positive controls. Two replicate cultures were prepared at each test point. Lymphocyte cultures were treated approximately forty-eight 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
The composition of the treatment media was as follows:
Test item solution 0.50mL
Culture medium (without PHA) 4.50mL
For the short treatment series, the treatment mediawere 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.

- Harvesting and slide preparation
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 waywere dropped onto clean,wet, greasefree 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.

- Slide evaluation
The cytokinesis-block proliferation index CBPI was calculated as follows:
CBPI = (mononucleated + 2×binucleated + 3×multinucleated) / total number of cells counted
where mononucleated, binucleated and multinucleated are respectively the number of mononucleated cells, binucleated cells and multinucleated cells. CBPI was 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 percentage cytotoxicity was evaluated according to the following formula:
%Cytotoxicity = 100−100*[(CBPI T −1)/(CBPI C −1)]
where:
T = test item treated culture
C = solvent control culture
The highest dose level for genotoxicity assessment was selected as a dose which produces a substantial cytotoxicity compared with the solvent control. Ideally the cytotoxicity should be between 50 % and 60 %. 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 Mitomycin-C, 1000 binucleated cells per cell culture were scored to assess the frequency of micronucleated cells.
Concerning cultures treated with Colchicine, since it is a known mitotic spindle poison which induces mitotic slippage and cytokinesis block, a greater magnitude of response was observed in mononucleated cells. For this reason, 1000 mononucleated cells per cell culture were scored.
The criteria for identifying micronuclei were as follows:
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

- Solubility test
Solubility of the test item was evaluated in a preliminary trial using complete culture medium.

- Osmolality and pH results
Following treatment with the test item, variations of pH or osmolality were monitored.

- Selection of dose levels for scoring
The CBPI was calculated for each of the treatment series.

Evaluation criteria:
In this assay, the test item 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 item 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 increasewhenevaluated with theCochran-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.
Species / strain:
lymphocytes: human whole body
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Solubility test
The test item gave an opaque formulation with heavy and moderate precipitation in culture medium at the concentrations of 50.0 and 25.0 mg/mL respectively, expressed as organic content. An opaque formulate without visible precipitation, feasible for treatment was obtained at 12.5 mg/mL. An opaque solution feasible for dilution was obtained at the concentration of 6.29 mg/mL.
On the basis of these results and the cytotoxicity results obtained in the MLA Study No. A3170, the maximum dose level of 1250 µg/mL was selected for the cytogenetic test by using a suspension at 12.5 mg/mL added to the treatment medium in the ratio 1:10.
Following treatment with the test item, no precipitate visible by eye was noticed at the beginning or by the end of treatment at any concentration tested.

- Osmolality and pH results
Following treatment with the test item, no remarkable variation of pH or osmolality was observed at any dose level, in the absence or presence of S9 metabolism.

- Selection of dose levels for scoring
Following the 3 hour treatment, no remarkable cytotoxicity was observed at any dose level.
Following the continuous treatment, a cytotoxicity of 65 % and 23 % respectively was calculated for the two highest dose levels of 1250 and 826 µg/mL, where precipitation of the test item, which interfered with the scoring of micronuclei, was seen onto slides.
Moderate cytotoxicity was seen at the lower dose level of 556 µg/mL (47 %), while no remarkable cytotoxicity was seen over the remaining dose range.
On the basis of the above results, the dose levels selected for scoring of micronuclei were as follows:
Short Treatment 1250, 826 and 556 with the respective cytotoxicity 18, 7 and 11 %
Long Treatment 556, 371 and 247 with the respective cytotoxicity 47, 18 and 15 %
For the positive control, the following dose levels were selected for scoring:
Short treament Mitomycin-C 0.500
Long treatment Colchicine 0.0800
Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controlsMitomycin-C and Colchicine, indicating the correct functioning of the test system. ForMitomycin-C the response was slightly out of the historical range (observed incidence 6% and range of incidence 1.60 - 5.35%). However, for both positive controls the results were compatible with those generated in our historical control database. The study was accepted as valid.
- Analysis of results
Adequate cell proliferation was observed in negative control cultures and the appropriate number of doses and cells was analysed.

Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment series.
All incidenceswere within the normal distribution of historical control data (95 % confidence limits) with the exception of the result obtained at the intermediate (short treatment) and high dose level (continuous treatment). These values slightly exceeded the upper confidence limit, but fell within the historical control range (0.0-1.05 and 0.1-1.20) and were consistent with those reported in literature for female donors (Fenech et al., 1994, Bonassi et al., 1995, Bonassi et al., 2001). No concentration related increase was seen, hence these results were considered without any biological relevance.
Conclusions:
The substance does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.
Executive summary:

The test item was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment, according to the OECD guideline 487 (2016).

Since the test item gave positive results in a mouse lymphoma mutation assay without any difference in the absence and presence of S9 metabolic activation, a direct genotoxic mechanism was suspected.

This study was intended as a further investigation to evaluate cytogenetic effects and treatments were carried out only in the absence of S9 metabolism.

Two treatment series were performed. A short termtreatment, where the cells were treated for 3 hours and the harvest time of approximately 32 hours, corresponding to approximately two cell cycle lengths was used and a long term (continuous), treatment where the cells were treated until harvest at 31 hours.

Solutions of the test item were prepared in complete culture medium.

On the basis of the solubility of the test item and cytotoxicity results obtained in the MLA test, the maximum dose level of 1250 µg/mL expressed as organic content or 1730 µg/mL expressed as test item as received, was selected for treatment. Lower dose levels of 826, 556, 371, 247, 165, 110, 73.2 and 48.8 µg/mL expressed as organic content or 1140, 769, 513, 342, 228, 152, 101 and 67.5 µg/mL expressed as test item as received, were chosen for

the short termtreatment. For the continuous treatment, the additional dose level of 32.5 µg/mL expressed as organic content or 45.0 µg/mL expressed as test item as received was included.

The experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point.

The actin polymerisation inhibitor Cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. The cytokinesisblock

proliferation index CBPI was calculated in order to evaluate cytotoxicity.

Dose levels for the scoring of micronuclei were selected with the aim to evaluate the test item concentrations at adequate levels of cytotoxicity, covering a range from the maximum

(55 ± 5%) to slight or no toxicity. In the absence of cytotoxicity, the maximum dose level was selected as the highest dose level for scoring.

Based on the results obtained, the following concentrations were selected for the scoring of micronuclei:

- short treatment: 1250, 826 and 556 µg/mL (organic content) with Cytotoxicity 18, 7 and 11 % respectively;

- continuous treatment: 556, 371 and 247 µg/mL (organic content) with Cytotoxicity 47, 18 and 15 % respectively.

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 controlsMitomycin-C and Colchicine, indicating the correct functioning of the test system. The study was accepted as valid.

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 (95% control limits) with the exception of the result obtained at the intermediate dose level (short treatment) and high dose level (continuous treatment). These values slightly exceeded the upper confidence limit, but fell within the historical control range and no concentration related increase was seen. Hence, this result was considered without any biological relevance.

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

Data source

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Results and discussion

Applicant's summary and conclusion