Reaction products of stearic acid with 2-aminoethanol, maleic anhydride and sodium sulphite

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

• Ames test (GLP OECD 471 guideline study, Kr.1): No evidence of mutagenic activity in the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvrA in the presence or the absence of S9-mix.
• In vitro micronucleus test (GLP OECD 497 guideline study, Kr.1): The registered substance did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, either in the presence or absence of a rat liver metabolizing system.

• In vitro Mammalian Cell assay (MLA) (GLP OECD 490 guideline study, Kr.1): A borderline effect of the test substance was observed in the presence or absence of a metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-02-20 - 2017-11-27

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:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

Test concentrations with justification for top dose:

Concentrations were selected on the basis of the Preliminary Solubility Test and Preliminary Range Finding Test (Informatory Toxicity Test). In the Initial Mutation Test and Confirmatory Mutation Test, the different concentrations were used.
Preliminary Compatibility Test
The solubility of the test item was examined using Distilled water, N,N-Dimethylformamide (DMF) and Dimethyl sulfoxide (DMSO). Partial dissolution was observed at 100 mg/mL concentration using DMSO and DMF. Test item was soluble at the same concentration using Distilled water after approximately 3 minutes vortex. Therefore, Distilled water was selected as vehicle (solvent) for the study.
Preliminary Concentration Range Finding Test (Informatory Toxicity Test)
Based on the solubility test, a 100 mg/mL stock solution was prepared in Distilled water. Seven test concentrations were prepared by successive dilutions of the stock solution, spaced by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98 and TA100) were determined at concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item, in the absence and presence of metabolic activation. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.
Test Item Concentrations in the Mutagenicity Tests (Initial Mutation Test and Confirmatory Mutation Test)
Based on the results of the preliminary tests, a 100 mg/mL stock solution was prepared in Distilled water. Maximum seven test concentrations were prepared by successive dilutions of the stock solution, to obtain lower doses. The maximum test concentration
was 5000 μg test item/plate. Examined concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate. Examined concentrations in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The solubility of the test item was examined using Distilled water, N,N-Dimethylformamide (DMF) and Dimethyl sulfoxide (DMSO). Partial dissolution was observed at 100 mg/mL concentration using DMSO and DMF. Test item was soluble at the same concentration using Distilled water after approximately 3 minutes vortex. Therefore, Distilled water was selected as vehicle (solvent) for the study.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water and DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylenediamine (NPD), 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.
- Cell density at seeding (if applicable): Not applicable
DURATION
- Preincubation period: 20 min at 37ºC
- Exposure duration: 48±1 hours at 37ºC
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A
SELECTION AGENT (mutation assays): N/A
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A
NUMBER OF REPLICATIONS: In the main tests, each sample (including the controls) was tested in triplicate.
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: N/A
NUMBER OF CELLS EVALUATED: N/A
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: N/A
DETERMINATION OF CYTOTOXICITY: N/A
OTHER EXAMINATIONS:
- Determination of polyploidy: N/A
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

Evaluation criteria:

The colony numbers on the untreated / negative (solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard deviation and the mutation factor* values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

Criteria for Validity:
The study was considered valid if:
- the number of revertant colonies of the negative (vehicle/solvent) and positive controls were in the historical control range in all strains of the main tests;
- at least five analyzable concentrations were presented in all strains of the main tests.

Criteria for a Positive Response:
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.

An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (so lvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.

Criteria for a Negative Response: The test item was considered to have shown no mutagenic activity in this study if it produces neither
a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Statistics:

No statistics performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

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:

no cytotoxicity

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)

In the Preliminary Range Finding Test, the plate incorporation method was used. The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester strains in the presence and absence of metabolic activation system (±S9 Mix) with appropriate untreated, negative (solvent) and positive controls.

Each sample (including the controls) was tested in triplicate.

Following concentrations were examined: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.

In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).

No precipitate was detected on the plates in the preliminary experiment.

Inhibitory or toxic effects of the test item were not detected in the preliminary experiment.

Based on the results of the Range Finding Test and the solubility findings, the maximum final concentration to be tested in the main experiments was 5000 μg/plate.

INITIAL AND CONFIRMATORY MUTATION TESTS

In the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used. The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and the Escherichia coli WP2 uvrA strain. The Initial Mutation Test and Confirmatory Mutation Test were

performed in the presence and absence of a metabolic activation system. Each test was performed with appropriate untreated, negative (solvent) and positive controls. In the main tests each sample (including the controls) was tested in triplicate.

Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate and in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

No precipitate was detected on the plates in the main tests in the examined bacterial strains with and without metabolic activation.

No inhibitory, cytotoxic effect of the test item was detected in the main tests in all examined bacterial strains with and without metabolic activation.

In the Initial Mutation Test and Confirmatory Mutation Test, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls. There were no reproducible dose-related trends and there was no indication of any treatment-related effect.

In the Initial Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1535 bacterial strain at 158.1 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.52). However, there was no dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the number of revertant colonies was within the historical control range.

In the Confirmatory Mutation Test (pre-incubation method), the highest revertant rate was observed in Escherichia coli WP2 uvrA bacterial strain at 15.81 μg/plate concentration without metabolic activation (the observed mutation factor value was: MF: 1.25). However, there was no dose-response relationship, the number of revertant colonies did not show any biologically relevant increase compared to the solvent controls and the number of revertant colonies was within the historical control range.

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.

Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

Conclusions:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a metabolic activation system, which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.
The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test and a Confirmatory Mutation Test. In the Preliminary Concentration Range Finding Test as well as in the Initial Mutation Test the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.
The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item Sopromine 1686 has no mutagenic activity in the bacterial strains under the test conditions used in this study.

Executive summary:

The test item, Sopromine 1686, was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavoneinduced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method). Batch 3900075 of Sopromine 1686 was a creamy white liquid with a purity of 25.1%. All concentrations and dose-levels were expressed as active ingredient content of the test item (a correction factor of 3.98 was used).

Distilled water at a concentration of 100 mg/mL. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test in tester strains Salmonella typhimurium TA100 and TA98 in the absence and presence of metabolic activation. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test were 5000, 1581, 500, 158.1, 50 and

15.81 μg/plate, in the Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

No precipitate was detected on the plates in the Preliminary Concentration Range Finding Tests and in the main tests in all examined bacterial strains with and without metabolic activation. No inhibitory, cytotoxic effect of the test item was detected in the Preliminary Range Finding Test or in the main tests. The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used .

In conclusion, the test item Sopromine 1686 (Batch Number: 3900075) has no mutagenic activity in the bacterial strains under the test conditions used in this

study.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 December 2017 to 10 July 2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: L5178Y TK+/- cells were obtained from ATCC (American Type Culture Collection, Manassas, USA), by the
intermediate of Biovalley (Marne-La-Vallée, France).
- Suitability of cells: recommended by international regulations for in vitro mammalian cell gene mutation test and for in vitro micronucleus test. Indeed, they are suitable to reveal chemically induced micronuclei.
- Methods for maintenance in cell culture if applicable: Cell cultures were grown at 37°C in a humidified atmosphere of 5% CO2/95% air in culture medium.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The culture medium was RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 μg/mL) and sodium pyruvate (200 μg/mL). This medium was supplemented by heat-inactivated horse serum at 10% (v/v).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (induced enzymatic systems contained in rat liver post-mitochondrial fraction)

Test concentrations with justification for top dose:

The highest dose level selected for the micronucleux analysis of the main experiments were based on the level of cytotoxicity reported in the preliminary experiment as well cell cytoxicity and precipitated reported in the main experiments.
Experiments without S9 mix:
The dose levels selected for micronucleus analysis were as follows:
- 12.5, 25 and 50 μg/mL for the 3-hour treatment, the latter being the lowest dose level showing precipitate in the culture medium at the end of the treatment period,
- 25, 37.5 and 50 μg/mL for the 24-hour treatment in the first experiment, the latter inducing a 29% decrease in the PD and the higher dose level being too cytotoxic,
- 12.5, 25 and 50 μg/mL for the 24-hour treatment in the second experiment, the higher dose levels being too cytotoxic.

Experiment with S9 mix:
The dose levels selected for micronucleus analysis were 12.5, 25 and 37.5 μg/mL, the latter being the lowest dose level showing precipitate in the culture medium at the end of the treatment period.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water for injections
- Justification for choice of solvent/vehicle: Available solubility data showed that the registered was soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension in medium
- Cell density at seeding (if applicable): On the day of treatment, cells were counted and suspended in order to reach approximately 3 x 105 cells/mL (final concentration = N0) in the final treatment medium (culture medium containing 5% inactivated horse serum).

DURATION
- Preincubation period: N/A
- Exposure duration: Without S9 mix: 3 h treatment + 24 h recovery and 24 h treatment + 0 h recovery / With S9 mix: 3 h treatment + 24 h recovery
- Expression time (cells in growth medium): see above
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): see above

SELECTION AGENT (mutation assays): N/A

SPINDLE INHIBITOR (cytogenetic assays): N/A

STAIN (for cytogenetic assays): The slides were stained for approximately 15 min in 5% Giemsa.

NUMBER OF REPLICATIONS: duplicate (two cultures/dose level)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were dropped onto clean glass slides. The slides were air-dried before being stained for approximately 15 min in 5% Giemsa

NUMBER OF CELLS EVALUATED: For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 1000 mononucleated cells per dose). Appropriate test item dose levels for scoring of micronuclei were selected mainly on the basis of the achieved reduction of PD and on the presence of precipitate.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): N/A

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
Analysis was performed by microscopic evaluation, on the basis of the recommendations of Miller et al. (1995), according to the following criteria:
- micronuclei should be located within the cytoplasma of the cell,
- micronuclei should be clearly surrounded by a nuclear membrane,
- micronuclei should be round or oval in shape,
- the micronucleus area should be less than one-third of the area of the main nucleus,
- micronuclei should be non-refractile (can be distinguished from artefacts such as staining particles),
- micronuclei should not be linked to the main nucleus via nucleoplasmic bridges,
- micronuclei should have similar staining intensity to that of the main nuclei,
- micronuclei may touch but not overlap the main nuclei and the micronuclear boundary should be distinguishable from the nuclear boundary,
- only mononucleated cells with a number of micronuclei ≤ 5 will be scored to exclude apoptosis and nuclear fragmentation.

DETERMINATION OF CYTOTOXICITY
- Method: For each culture, the Population Doubling (PD) (d) was calculated and used relative to that of the vehicle control. The population doubling is the log of the ratio of the final count at the time of harvesting (N) to the starting count (N0), divided by the log of 2.
- Any supplementary information relevant to cytotoxicity: The cytotoxicity induced by a treatment was evaluated by the decrease in the PD, when compared to the vehicle control (Mean % PD of the vehicle control set to 100%).

OTHER EXAMINATIONS:
- Determination of polyploidy: no
- Determination of endoreplication: No
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

Evaluation criteria:

The biological relevance of the results was always taken into account when evaluating results.

Evaluation of a positive response: a test item is considered to have clastogenic and/or aneugenic
potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was demonstrated by a statistically significant trend test,
- for at least one dose level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose levels.

Evaluation of a negative response: a test item is considered clearly negative if none of the criteria for a positive response was met.

Statistics:

For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures.
This comparison was performed using the x2 test, unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 was used as the lowest level of significance. This statistical analysis was performed using a validated Excel sheet.
To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose levels. This statistical analysis was performed using SAS Enterprise Guide software.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not assessed
- Effects of osmolality: Not assessed
- Evaporation from medium: Not assessed
- Water solubility: According to available solubility data, the vehicle used for the preparation of test item dose formulations and the treatment of vehicle control cultures was water for injections.
- Precipitation: In the preliminary test, a precipitate was observed in the culture medium at the end of the treatment period, at dose levels ≥ 138.9 µg/mL
- Definition of acceptable cells for analysis: see section "Method"
- Other confounding effects: N/A

RANGE-FINDING/SCREENING STUDIES:
Based on available solubility data, the highest dose level selected for the preliminary cytotoxicity test was 1250 µg/mL, since it was expected to produce precipitation in the culture medium. This dose level was obtained using a test item stock formulation at the concentration of 50 mg/mL and a treatment volume of 2.5% (v/v) in the culture medium.
The dose levels selected for the treatment of the preliminary test were 1.715, 5.144, 15.43, 46.30, 138.9, 416.7 and 1250 µg/mL.
At the highest dose level of 1250 µg/mL, the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was 297 mOsm/kg H2O (295 mOsm/kg H2O for the vehicle control). Therefore, none of the tested dose levels was considered to produce extreme culture conditions.
A precipitate was observed in the culture medium at the end of the treatment period, at dose levels ≥ 138.9 µg/mL.
Following the 3-hour treatments with and without S9 mix, a severe cytotoxicity was observed at dose levels ≥ 138.9 µg/mL, as shown by a 100% decrease in the PD.
Following the 24-hour treatment without S9 mix, a marked to severe cytotoxicity was observed at dose levels ≥ 46.30 µg/mL, as shown by a 67 to 100% decrease in the PD


NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: see details in section below
- Indication whether binucleate or mononucleate where appropriate: see details in section below

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see Tables 1.1 and 1.2. Positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells
- Negative (solvent/vehicle) historical control data: see Tables 1.1 and 1.2. No statistically significant increase in the frequency of micronucleated cells was noted relative to the vehicle controls, either after the 3- or the 24-hour treatment. Frequencies of micronucleated cells remained consistent with vehicle control historical data and no dose response relationship was demonstrated by the linear regression (p>0.05).

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: The cytotoxicity induced by a treatment was evaluated by the decrease in the Population Doubling (PD), when compared to the vehicle control (Mean % PD of the vehicle control set to 100%).
- Other observations when applicable: N/A

Historical data:

Table 1.1 :Experiments without S9 mix :

Parameter	Frequency of micronucleated cells in 1000 cells
Treament conditions	3 hours treatment + 24 hours recovery			24 hours treatment + 0 hours recovery
Control items	Vehicle control	MMC (1 µg/mL)	COL (0.5 µg/mL)	Vehicle control	MMC (1 µg/mL)	COL (0.5 µg/mL)
n	50	50	50	28	28	28
Mean	1.7	133.0	22.9	2.1	46.4	34.5
SD	1.0	67.5	13.3	1.1	19.8	17.5
Lower CL 95%	1.4	113.8	19.1	1.6	38.7	27.7
Upper CL 95%	1.9	152.2	26.6	2.5	54.1	41.2

 

Table 1.2 :Experiments with S9 mix :

Parameter	Frequency of micronucleated cells in 1000 cells
Treament conditions	3 hours treatment + 24 hours recovery
Control items	Vehicle control	CPA (6 µg/mL)
n	67	67
Mean	1.5	101.9
SD	0.9	49.3
Lower CL 95%	1.3	89.8
Upper CL 95%	1.7	113.9

COL: Colchicine

MMC: Mitomycin c

CL: Confidence limit

Max: Maximal value

CPA: Cyclophosphamide

SD: Standard deviation

Min: Minimal value

n: number of values

Detailed results on the main experiments:

Experiments without S9 mix

No statistically significant increase in the frequency of micronucleated cells was noted relative to the vehicle controls, either after the 3- or the 24-hour treatment. Frequencies of micronucleated cells remained consistent with vehicle control historical data and no dose‑response relationship was demonstrated by the linear regression (p>0.05).

These results met the criteria for a negative response.

Experiment with S9 mix

No statistically significant increase in the frequency of micronucleated cells was noted relative to the vehicle control. At the dose level of 37.5 µg/mL, the frequency of micronucleated cells of each replicate culture was slightly above the vehicle control historical range (5‰ and 4‰ versus [0 to 3.5‰] for the historical data). Since no statistical significance was observed, since no dose-response relationship was demonstrated by the linear regression (p>0.05 ) and since the mean frequency of micronucleated cells remained < 5‰, this slight increase was considered as non biologically relevant and the overall results were considered to show a negative response.

Conclusions:

Under the experimental conditions of the study, the test item, Sopromine 1686, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system

Executive summary:

The objective of this study was to evaluate the potential of the test item, Sopromine 1686, to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178Y TK+/-.

Methods:

After a preliminary cytotoxicity test, the test item Sopromine 1686, diluted in water for injections, was tested in two independent cytogenetic experiments with or without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

First experiment: 3 treatment + 24 h recovery and 24 h treatment + 0 h recovery

Second experiment: 24 h treatment + 0 h recovery

With S9 mix: 3 h treatment + 24 h recovery

Each treatment was coupled to an assessment of cytotoxicity at the same dose levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells.

After the final cell counting, the cells were washed and fixed. Then, cells from at least three dose levels of the test item-treated cultures were dropped onto clean glass slides. The slides were air-dried before being stained in 5% Giemsa. Slides from vehicle and positive controls cultures were also prepared as described above. All slides were coded before analysis, so that the analyst was unaware of the treatment details of the slide under evaluation ("blind" scoring). For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose levels of the test item, for the vehicle and the positive controls, in

1000 mononucleated cells per culture (total of 2000 mononucleated cells per dose).

Number of cells with micronuclei and number of micronuclei per cell were recorded separately for each treated and control culture.

Results:

Since the test item was found to be cytotoxic in the preliminary test, the highest dose level selected for the main experiments was based on the level of cytotoxicity, according to the criteria specified in the international regulations.

The mean Population Doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be

valid.

Experiments without S9 mix:

With a treatment volume of 2.5% (v/v) in culture medium, the dose levels selected for the treatments were as follows:

- 6.25, 12.5, 25, 37.5, 50, 75, 100 and 150 μg/mL for the 3-hour treatment (first experiment),

- 3.13, 6.25, 12.5, 18.8, 25, 37.5, 50 and 100 μg/mL for the 24-hour treatment in the first experiment,

- 6.25, 12.5, 25, 37.5, 50, 66.7, 75 and 100 μg/mL for the 24-hour treatment in the second experiment.

A precipitate was observed in the culture medium at the end of the treatment periods, at dose levels ≥ 50 μg/mL.

Cytotoxicity:

Following the 3-hour treatment, a moderate to severe cytotoxicity was induced at dose levels ≥ 75 μg/mL, as shown by a 47 to 100% decrease in the PD.

Following the 24-hour treatment, a slight to severe cytotoxicity was induced at dose levels ≥ 50 μg/mL, as shown by a 29 to 100% decrease in the PD in the first experiment, and a marked to severe cytotoxicity was induced at dose levels ≥ 66.7 μg/mL, as shown by a 68 to 100% decrease in the PD in the

second experiment.

Micronucleus analysis:

The dose levels selected for micronucleus analysis were as follows:

- 12.5, 25 and 50 μg/mL for the 3-hour treatment, the latter being the lowest dose level showing precipitate in the culture medium at the end of the treatment period,

- 25, 37.5 and 50 μg/mL for the 24-hour treatment in the first experiment, the latter inducing a 29% decrease in the PD and the higher dose level being too cytotoxic,

- 12.5, 25 and 50 μg/mL for the 24-hour treatment in the second experiment, the higher dose levels being too cytotoxic.

No statistically significant increase in the frequency of micronucleated cells was noted relative to the vehicle controls, either after the 3- or the 24-hour treatment. Frequencies of micronucleated cells remained consistent with vehicle control historical data and no dose-response relationship was demonstrated by the linear regression (p>0.05). These results met the criteria for a negative response.

Experiment with S9 mix:

With a treatment volume of 2.5% (v/v) in culture medium, the dose levels selected for the treatment were 6.25, 12.5, 25, 37.5, 50, 75, 100 and 150 μg/mL.

A precipitate was observed in the culture medium at the end of the treatment period, at dose levels ≥ 37.5 μg/mL.

Cytotoxicity:

A slight to severe cytotoxicity was induced at dose levels ≥ 75 μg/mL as shown by a 31 to 100% decrease in the PD.

Micronucleus analysis:

The dose levels selected for micronucleus analysis were 12.5, 25 and 37.5 μg/mL, the latter being the lowest dose level showing precipitate in the culture medium at the end of the treatment period.

No statistically significant increase in the frequency of micronucleated cells was noted relative to the vehicle control. At the dose level of 37.5 μg/mL, the frequency of micronucleated cells of each replicate culture was slightly above the vehicle control historical range (5‰ and 4‰ versus [0 to 3.5‰] for the

historical data). Since no statistical significance was observed, since no dose-response relationship was demonstrated by the linear regression (p>0.05) and since the mean frequency of micronucleated cells remained < 5‰, this slight increase was considered as non biologically relevant and the overall results

were considered to show a negative response.

In conclusion, under the experimental conditions of the study, the test item, Sopromine 1686, did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or absence of a rat liver metabolizing system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 17 April to 20 November 2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: L5178Y TK+/- cells were obtained from ATCC (American Type Culture Collection, Manassas, USA)
- Suitability of cells: recommended by international regulations.
- Methods for maintenance in cell culture if applicable: For each experiment, one or more vials was thawed rapidly, cells were diluted in RPMI-10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: The culture medium was RPMI 1640 medium containing 10% v/v Horse serum (heat inactivated), 0.01 mL/mL Antibiotic-antimycotic solution, 0.5 mg/mL Pluronic-F68 and 0.2 mg/mLPyruvic acid.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (induced enzymatic systems contained in rat liver post-mitochondrial fraction)

Test concentrations with justification for top dose:

Treatment concentrations for the mutation assays were selected on the basis of the result of a short preliminary toxicity test. Three-hour treatment in the presence and absence of S9-mix and 24-hour treatment in the absence of S9-mix was performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest concentration tested in the preliminary test was 2 mg/mL
Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:
Assay 1, 3-hour treatment with metabolic activation: 150, 125, 100, 75, 50, 25, 12.5 and 6.25 μg/mL,
Assay 1, 3-hour treatment without metabolic activation: 120, 100, 80, 60, 40, 20, 10 and 5 μg/mL,
Assay 2, 3-hour treatment with metabolic activation: 150, 125, 100, 75, 50, 25, 12.5 and 6.25 μg/mL,
Assay 2, 3-hour treatment without metabolic activation: 150, 120, 100, 80, 60, 40, 20, 10 and 5 μg/mL,
Assay 2, 24-hour treatment without metabolic activation: 150, 120, 100, 80, 60, 40, 20, 10, 5 and 2.5 μg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water was used for vehicle of the test item. Dimethyl sulfoxide was used for vehicle (solvent) of the positive control chemicals.
- Justification for choice of solvent/vehicle: Available solubility data showed that the registered substance was soluble in water.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension in medium
- Cell density at seeding (if applicable): For the 3-hour treatments, 10e7 cells were placed in each of a series of 75 cm2 sterile flasks. For the 24-hour treatment, 6x10e6 cells were placed in each of a series of 25 cm2 sterile flasks. treatment medium contained a reduced serum level of 5% (v/v) RPMI-5.

DURATION
- Preincubation period: N/A
- Exposure duration: Without S9 mix: 3 h treatment and 24 h treatment / With S9 mix: 3 h treatment
- Expression time (cells in growth medium between treatment and selection): To allow expression of TK- mutations, cultures were maintained in flasks for 2 days. During the expression period, sub-culturing was performed daily. On each day, cell density was adjusted to a concentration of 2x10e5 cells/mL (whenever possible) and transferred to flasks for further growth.
- Selection time (if incubation with a selective agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A
- Method used: microwell plates.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure: At the end of the expression period, the cell concentration was adjusted to 1x10e4 cells/mL. Trifluorothymidine (TFT) (300 μg/mL stock solution) was diluted 100-fold into these suspensions to give a final concentration of 3 μg/mL. Using a multi-channel pipette, 0.2 mL of each suspension was placed into each well of four, 96-well microplates (384 wells) at 2x10e3 cells per well. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks (12 days) and wells containing clones were identified by eye and counted. In addition, scoring of large and small colonies was performed to obtain information on the possible mechanism of action of the test item, if any.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative survival (RS), relative total growth (RTG) and Suspension Growth (SG).:

METHODS FOR MEASUREMENTS OF GENOTOXICIY: Determination of Mutant Frequency: In order to calculate the MF ("mutants per 10e6 viable cells"), the plating efficiencies of both mutant and viable cells in the same culture were calculated as follows: MF = [P (mutant)/2 x 103] x [1.6/P (viable)] x 10e6 = {-ln [EW/TW (mutant)]/-ln [EW/TW (viable)]} x 800

OTHERS: ASSAY ACCEPTANCE CRITERIA
The assay was considered valid if all of the following criteria were met (based on the relevant guidelines):
1. The mutant frequency in the negative (vehicle) control cultures fell within the normal range (50-170 mutants per 10e6 viable cells).
2. The positive controls met at least one of the following two criteria:
-The positive control chemical demonstrated an absolute increase in total MF that is, an increase above the spontaneous background MF of at least 300 x 10-6. At least 40% of the IMF reflected in the small colony MF.
-The positive control substance had an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent untreated/vehicle control (a small colony IMF of 150 x 10-6).
3. The plating efficiency (PEviability) of the negative (vehicle) controls was within the range of 65% to 120% at the end of the expression period.
4. At least four test concentrations were present, where the highest concentration produced approximately 80-90% toxicity (measured by RTG), resulted in precipitation, or it was 2 mg/mL, 2 μL/mL or 0.01 M (whichever is the lowest), or it was the highest practical (achievable) concentration.

Evaluation criteria:

The test item was considered to be clearly positive (mutagenic) in this assay if all the following criteria were met:
1. At least one concentration exhibited a statistically significant increase (p<0.05) compared with the concurrent negative (vehicle) control and the increase was biologically relevant (i.e. the mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 10e6 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle) control value).
2. The increases in mutation frequency were reproducible between replicate cultures and/or between tests (under the same treatment conditions).
3. The increase was concentration-related (p < 0.05) as indicated by the linear trend analysis.

The test item was considered clearly negative (non-mutagenic) in this assay if in all experimental conditions examined there was no concentration related response or, if there is an increase in MF, but it did not exceed the GEF. Then, test item was considered unable to induce mutations in this test system.

Statistics:

Statistical significance of mutant frequencies (total wells with clones) was performed using Microsoft Excel software.

The negative (vehicle) control log mutant frequency (LMF) was compared to the LMF of each treatment concentration, based on Dunnett's test for multiple comparisons and the data were checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend was one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No change in pH detected
- Data on osmolality: No change in osmolality detected
- Possibility of evaporation from medium: No
- Water solubility: distilled water was selected as the vehicle of the study.
- Precipitation and time of the determination: N/A
- Definition of acceptable cells for analysis: N/A

RANGE-FINDING/SCREENING STUDIES:
Insolubility and excessive cytotoxicity was detected in the preliminary experiment. Precipitate was detected in the 2000-500 μg/mL concentration range in case of short treatment with and without metabolic activation and in the 2000-250 μg/mL concentration range in case of long treatment without metabolic activation. Minimal amount of precipitate was also recorded at some lower concentrations (down to 125 μg/mL in case of the short treatment with metabolic activation, 3.906 μg/mL in case of the short treatment without metabolic activation and 31.25 μg/mL in case of the long treatment without metabolic activation). Excessive cytotoxicity was also detected in the preliminary experiments: no cells survived the expression period in the 2000-250 μg/mL concentration range using the short treatment with and without metabolic activation and in the 2000-125 μg/mL concentration range using the long treatment without metabolic activation.
Concentrations up to the cytotoxic range were selected for the main experiments according to the instructions of the relevant OECD guideline. At least eight concentrations were selected for the main experiments in each assay.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
The positive controls (Cyclophosphamide in the presence of metabolic activation and 4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays (see historical control data below). All of the positive control samples in the performed experiments fulfilled at least one of the relevant OECD criteria.
The plating efficiencies for the negative (vehicle) controls of the test item and positive control item as well as the untreated control sample at the end of the expression period (PEviability) were within the acceptable range (65-120%) in all assays.

- RESULTS OF ASSAYS 1 & 2 (see summary table):
- In Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Treatment concentrations were 150, 125, 100, 75, 50, 25, 12.5 and 6.25 μg/mL (experiment with metabolic activation); and 120, 100, 80, 60, 40, 20, 10 and 5 μg/mL (experiment without metabolic activation).
In the presence of S9-mix (3-hour treatment), marked cytotoxicity of the test item was observed. An evaluation was made using data of all concentrations (relative total growth of the highest evaluated concentration of 150 μg/mL was 19%). Statistically significant increases in the mutation frequency were noted at 150 and 125 μg/mL concentrations, but the difference between those values and the negative (vehicle) control data did not exceed the Global Evaluation Factor, thus was not biologically fully relevant. A dose-response relationship was also observed by linear trend analysis. Overall, this experiment was not considered to fulfil all the criteria of positivity (borderline).
In the absence of S9-mix (3-hour treatment), marked cytotoxicity of the test item was observed (survival results are detailed in Table 2 of Appendix 5). An evaluation was made using data of all concentrations (relative total growth of the highest evaluated concentration of 120 μg/mL was 30%). Statistically significant increases in the mutation frequency were noted at 120 and 100 μg/mL concentrations, but the difference between those values and the negative (vehicle) control data remained well below the Global Evaluation Factor, thus those differences were considered as biologically not relevant. A significant dose-response to the treatment was also indicated by the linear trend analysis. This experiment was considered not to fulfil all the criteria of positivity (borderline), although the degree of cytotoxicity at the highest evaluated concentration was slightly lower than suggested by the OECD 490 guideline.

- In Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix), a 3-hour treatment without metabolic activation (in the absence of S9-mix) and a 24-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Based on the results of Assay 1, treatment concentrations were 150, 125, 100, 75, 50, 25, 12.5 and 6.25 μg/mL (experiment with metabolic activation); 150, 120, 100, 80, 60, 40, 20, 10 and 5 μg/mL (short treatment without metabolic activation) and 150, 120, 100, 80, 60, 40, 20, 10, 5 and 2.5 μg/mL (long treatment without metabolic activation.
In the presence of S9-mix (3-hour treatment), similarly to the first assay, excessive cytotoxicity of the test item (relative total growth of 6%) was observed at 150 μg/mL concentration. An evaluation was made using the data of the next concentration of 125 μg/mL (relative total growth of 17%) and the following six concentrations (a total of seven concentrations). Statistically significant increases in the mutation frequency were seen at 125 and 100 μg/mL concentrations, the increase was biologically relevant at 100 μg/mL concentrations (as the difference to the vehicle control was larger than the Global Evaluation Factor). A significant dose-response to the treatment was indicated by the linear trend analysis. However, there was no clear reproducibility between cultures (MF value of 387.0 was detected for replicate A, while MF value of 146.0 was detected for replicate B). Furthermore, the results were not reproducible at this concentration when compared to Assay 1 experiment with metabolic activation, therefore (mean values of 126.3 in Assay 1, 245.7 in Assay 2), this experiment was also considered as borderline.
In the absence of S9-mix (3-hour treatment), which was the repeated test of the experiment of Assay 1 using a modified concentration range to provide more adequate cytotoxicity data, marked cytotoxicity of the test item was observed. An evaluation was made using data of all concentrations (relative total growth of the highest evaluated concentration of 150 μg/mL was 12%). A statistically significant increase in the mutation frequency was noted at the 150 μg/mL concentration, but the difference between the observed value and the negative (vehicle) control data remained well below the Global Evaluation Factor, thus the observed effect was considered as biologically not fully relevant. A significant dose-response to the treatment was indicated by the linear trend analysis. This experiment was not considered to fulfil all the criteria of positivity (borderline).
In the absence of S9-mix (24-hour treatment), excessive cytotoxicity of the test item was observed, no cells survived the expression period in the 150, 120 and 100 μg/mL concentration samples. Excessive cytotoxicity (relative total growth of 3%) was also observed at 80 μg/mL concentration, while marked cytotoxicity was also recorded in the lower concentrations. An evaluation was made using data of the 60 μg/mL concentration (relative total growth of 7%) and the next five concentrations (a total of six concentrations). A statistically significant increase in the mutation frequency was noted at 60 μg/mL concentration, but similarly to experiments using the short treatment without metabolic activation, the difference between the observed value and the negative (vehicle) control data remained well below the Global Evaluation Factor, thus the observed effect was considered as biologically not fully relevant. A significant dose-response to the treatment was indicated by the linear trend analysis. This experiment was not considered to fulfil the criteria of positivity, and as the observed statistically significant increase was observed only at high degree of cytotoxicity (RTG<10%), based on the instruction of the relevant OECD 490 guideline, this experiment was considered as negative.
Results of the two main assays are summarized in the table below.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data) : see table below.

Summary of the results of Assays 1 and 2:

Assay	Concentration (RTG) and MF values	Increase in MF compared to vehicle control (GEF)	Dose response	Reproducible between cultures	Conclusion
Assay 1 3-hour +S9	150 μg/mL (19%): 179.1* 125 μg/mL (26%): 162.4* 100 μg/mL (32%): 126.3 Vehicle (100%): 84.9	94.2 (<GEF) 77.5 (<GEF) 41.4 (<GEF)	Yes	Yes	Borderline
Assay 2 3-hour +S9	125 μg/mL (17%): 192.8* 100 μg/mL (22%): 245.7* 75 μg/mL (62%): 103.7 Vehicle (100%): 97.7	95.1 (<GEF) 148.0 (>GEF) 6.0 (<GEF)	Yes	No	Borderline (lack of reproducibility between cultures)
Assay 1 3-hour -S9	120 μg/mL (30%): 120.6* 100 μg/mL (36%): 121.6* 80 μg/mL (62%): 100.9 Vehicle (100%): 59.1	61.5 (<GEF) 62.5 (<GEF) 41.8 (<GEF)	Yes	Yes	Borderline
Assay 2 3-hour -S9	150 μg/mL (12%): 175.1* 120 μg/mL (27%): 89.6 100 μg/mL (38%): 104.9 Vehicle (100%): 77.9	97.2 (<GEF) 11.7 (<GEF) 27.0 (<GEF)	Yes	Yes	Borderline
Assay 2 24-hour -S9	60 μg/mL (7%): 164.5* 40 μg/mL (60%): 94.5 20 μg/mL (127%): 84.2 Vehicle (100%): 89.1	75.4 (<GEF) 5.4 (<GEF) NA	Yes	Yes	Negative (Increase only at low RTG%, thus excessive cytotoxicity)

Notes: Results of the three highest evaluated concentrations are shown in the table. Statistical significance labelled by *. MF: Mutation Frequency (refers to 106 viable cells), GEF: Global Evaluation Factor (=126 per 10e6 viable cells), NA: Not Applicable

Historical data:

Mutation Frequency of the Negative Controls (2006-2016)
	Culture medium			Distilled water
Treatments	3h,S9 +	3h,S9 -	24h,S9 -	3h,S9+	3h,S9-	24h,S9-
Average SD Min. Max. n	94.3 26.9 39.3 198.5 84	103.6 35.3 52.6 235.6 43	106.4 27.4 41.7 179.1 44	90.4 22.7 33.4 121.8 26	96.6 19.0 55.1 125.0 13	96.3 24.6 43.2 141.1 13
	Dimethyl sulfoxide (DMSO)
Treatments	3h,S9 +	3h,S9 -	24h,S9 -
Average SD Min. Max. n	97.3 33.7 44.2 269.9 101	97.3 38.5 33.7 261.6 57	98.9 26.9 47.1 159.4 50
	Mutation Frequency of the Positive Controls (2006-2016)
	Cyclophosphamide			4-Nitroquinoline-N-oxide
Treatments	3h,S9+				3h,S9-	24h,S9-
Average SD Min. Max. n	1178.7 524.7 196.1 2642.5 106				722.2 330.0 223.5 1687.3 58	831.9 337.2 245.0 1577.6 52

 

Conclusions:

In conclusion, a borderline effect of Sopromine 1686 was observed in the presence or absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of Sopromine 1686 test item to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix). The design of this study was based on the OECD No. 490 guideline, and the study was performed in compliance with Citoxlab Hungary standard operating procedures and with the OECD Principles of Good Laboratory Practice.

Distilled water was used as the vehicle for the test item in this study. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:

Assay 1, 3-hour treatment with metabolic activation: 150, 125, 100, 75, 50, 25, 12.5 and 6.25 μg/mL,

Assay 1, 3-hour treatment without metabolic activation: 120, 100, 80, 60, 40, 20, 10 and 5 μg/mL,

Assay 2, 3-hour treatment with metabolic activation: 150, 125, 100, 75, 50, 25, 12.5 and 6.25 μg/mL,

Assay 2, 3-hour treatment without metabolic activation: 150, 120, 100, 80, 60, 40, 20, 10 and 5 μg/mL,

Assay 2, 24-hour treatment without metabolic activation: 150, 120, 100, 80, 60, 40, 20, 10, 5 and 2.5 μg/mL.

In Assays 1 and 2, there were no large changes in pH or osmolality after treatment. No insolubility was observed in the final treatment medium at the end of the treatment in any cases.

In Assay 1 and 2, following a 3-hour treatment with metabolic activation, excessive or marked cytotoxicity was seen in the higher concentration range. There were statistically significant increases in the mutation frequency values in the evaluated concentration range (at an acceptable degree of cytotoxicity) and a dose response was seen in both cases, however the difference between the calculated values and the control did not exceed the Global Evaluation Factor, GEF (thus showing no biological relevance), except in one case in Assay 2 at 100 μg/mL concentration. However, at that concentration no clear reproducibility was seen between replicates, and the effect was not reproducible between assays. Therefore, the experiments in the presence of metabolic activation system were considered not showing a consistent positive effect, but being borderline.

In Assay 1 and 2, following a 3-hour treatment without metabolic activation or 24-hour treatment without metabolic activation, excessive or marked cytotoxicity was seen in the higher concentration range. There were statistically significant increases in the mutation frequency values of the evaluated concentration range, although in case of the long treatment it could be seen only at a high degree (excessive level) of cytotoxicity. Dose response was seen in all cases, however the difference between the calculated values and the control did not exceed the Global Evaluation Factor, thus showing no biological relevance in all cases. Therefore, the experiments in the absence of metabolic activation system were considered as being borderline for the short treatment and negative for the long treatment.

The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.

The overall results clearly do not allow a conclusion of negative to be made, with reproducible results in more than one testing condition, that show statistical increases which fall below the regulatory cut-off (GEF) for a positive classification. Hence the overall conclusion needs to be that the test item is a borderline case.

In conclusion, a borderline effect of Sopromine 1686 was observed in the presence or absence of a metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Three GLP-compliant in vitro tests of Klimish reliability 1 are available to assess the genotoxic potential of the registered substance and were selected as key studies (CIToxLAB, 2018).

1) In an Ames test conducted according to OECD TG 471, the registered substance, formulated in distilled water, did not induce gene mutations by base pair changes or frameshifts in the genome of Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA in the presence and absence of a exogenous metabolic activation system (S9 fraction) at any dose levels (up to 5000 μg/plate). The substance Sopromine 1686 is therefore considered not to be mutagenic in this Ames test.

2) In an in vitro micronucleus test conducted according to OECD TG 487 using L5178Y TK+/- mouse lymphoma cells, the registered substance, formulated in distilled water, did not induce any statistically significant increase in the frequency of micronucleated cells when compared with the

vehicle controls, either after the 3- or the 24-hour treatment, in the presence or absence of a rat liver metabolizing system.

3) In a in vitro Mouse Lymphoma Assay conducted according to OECD TG 490 using the mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus, the registered substance, formulated in distilled water, induced a borderline effect following a 3-hour treatment with or without metabolic activation and was negative following a 24-hour treatment without metabolic activation.

Justification for classification or non-classification

The substance had no mutagenic activity in a Ames test and did not induce any significant increase in the frequency of micronucleated cells in a in vitro micronucleus test, in the presence and absence of a exogenous metabolic activation system. In a in vitro Mouse Lymphoma Assay, the registered substance induced a borderline effect following a 3-hour treatment with or without metabolic activation and was negative following a 24-hour treatment without metabolic activation. Based on these results, the registered substance is not classified for genotoxicty according to GHS and CLP regulations.