Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: non mutagenic (OECD 471, GLP, K, rel. 1).

Micronucleus test in human lymphocytes: non clastogenic (OECD 487, GLP, K, rel. 1).

HPRT test in Chines hamster lung fibroblasts cells (V79): non mutagenic (OECD 476, GLP, K, rel. 1)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 November - 31 December 2015
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
His+ for S. typhimurium
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: see table 7.6.1/1
Metabolic activation:
with and without
Metabolic activation system:
10% (v/v) S9 mix; S9 fraction prepared from liver homogenates of rats induced with Aroclor 1254 (500 mg/kg bw) by intraperitoneal route
Test concentrations with justification for top dose:
PRELIMINARY TOXICITY TEST:
- Test concentrations: 0 (vehicle: DMSO), 10, 100, 500, 1000, 2500 and 5000 μg/plate, with and without S9 mix in TA 98, TA 100 and TA 102 strains (direct plate incorporation method)
- Justification for top dose: Using a test item concentration of 100 mg/mL in the vehicle and a treatment volume of 50 μL/plate, the highest recommended dose-level of 5000 μg/plate was achievable. Thus, the top dose selected for the preliminary test was 5000 μg/plate.

MUTAGENICITY TESTS:
- Test concentrations without S9 mix
Experiment 1: 0, 0.8, 2.5, 7.4, 22.2, 66.7 and 200 μg/plate in all 5 strains (direct plate incorporation method)
Experiment 2: 0, 2.5, 7.4, 22.2, 66.7, 200 and 600 μg/plate in all 5 strains (direct plate incorporation method)
- Test concentrations with S9 mix:
Experiment 1: 0, 312.5, 625, 1250, 2500 and 5000 μg/plate for the TA 1535, TA 1537, TA 98 and TA 102 strains; 0, 156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate for the TA 100 strain (direct plate incorporation method)
Experiment 2: 0, 156.3, 312.5, 625, 1250, 2500 and 5000 μg/plate for the TA 102 strain; 0, 19.53, 39.06, 78.13, 156.3, 312.5 and 625 μg/plate for the TA 1535, TA 1537 and TA 98 strains; 0, 6.9, 20.6, 61.7, 185.2, 555.6, 1666.7 and 5000 μg/plate for the TA 100 strain (pre-incubation method)
- Justification for top dose: Since the test item was found freely soluble but toxic in the preliminary test, mainly in the absence of S9 mix, the selection of the highest dose-level to be used in the main experiments was based on the level of toxicity.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Vehicle was selected based on the available solubility data.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Anthramine
Remarks:
with metabolic activation
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: All five strains of Salmonella typhimurium were supplied by Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) or Culture Collections (Public Health England, Porton Down, Salisbury, SP4 0JG, UK).

METHOD OF APPLICATION: In agar (direct plate incorporation and pre-incubation method)

DURATION
- Preincubation period: 60 minutes at 37 °C
- Incubation period: 48-72 h at 37 °C for both direct plate incorporation and pre-incubation methods

NUMBER OF REPLICATIONS:
- Preliminary experiment: 1 plate/dose
- Main experiments: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- After 48 to 72 hours of incubation at 37°C, the number of revertants per plate was scored for each strain and for each experimental point using an automatic counter (Sorcerer Automatic Colony Counter for the scoring of colonies and Ames Study Manager for the data management, Perceptive Instruments Ltd, Bury St Edmunds IP33 3TA, UK). Also, the thinning of the bacterial lawn and the presence of precipitate were evaluated.
Rationale for test conditions:
Rationale for test concentrations:
- Preliminary toxicity test: Using a test item concentration of 100 mg/mL in the vehicle and a treatment volume of 50 μL/plate, the highest recommended dose-level of 5000 μg/plate was achievable. Thus, the top dose selected for the preliminary test was 5000 μg/plate.
- Main tests: Since the test item was found freely soluble but toxic in the preliminary test, mainly in the absence of S9 mix, the selection of the highest dose-level to be used in the main experiments was based on the level of toxicity.
Evaluation criteria:
In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

The test item was considered to have shown mutagenic activity in this study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls was observed, in any strain, at any dose-level,
- and/or a reproducible dose-response relationship was evidenced.

The test item was considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, was observed at any of the tested dose-levels,
- nor any evidence of a dose-response relationship was noted.
Statistics:
None
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
refer 'Additional information on results'
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
- Using a test item concentration of 100 mg/mL in the vehicle and a treatment volume of 50 μL/plate, the highest recommended dose-level of 5000 μg/plate was achievable.
- No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.
- A moderate to strong toxicity (decrease in the number of revertants and/or thinning of the bacterial lawn) was noted at dose-levels ≥ 100 μg/plate towards the three strains used in the absence of S9 mix.
- In the presence of S9 mix, a strong toxicity was noted at 5000 μg/plate in the TA 98 and TA 100 strains, whereas no noteworthy toxicity was noted in the TA 102 strain.

MUTAGENICITY TESTS:
- No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.
- Without S9 mix: In the first experiment, only a slight toxicity (thinning of the bacterial lawn) was noted at the highest dose-level of 200 μg/plate in all the tested strains. In the second experiment, a moderate to strong toxicity was noted at dose-levels ≥ 66.7 μg/plate in the TA 1535, TA 1537 and TA 100 strains, and ≥ 200 μg/plate in the TA 98 and TA 102 strains.
- With S9 mix: In the first experiment, using the direct plate incorporation method, a moderate to strong toxicity was noted at dose-levels ≥ 2500 μg/plate in the TA 1535, TA 1537 and TA 100 strains, whereas no noteworthy toxicity was noted in the TA 98 and TA 102 strains. In the second experiment, using the pre-incubation method, a moderate to strong toxicity was noted at dose-levels ≥ 312.5 μg/plate in the TA 1535, TA 1537 and TA 98 strains, ≥ 555.6 μg/plate in the TA 100 strain and ≥ 1250 μg/plate in the TA 102 strain.
- The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment, with or without S9 mix. These results with and without S9 mix met the criteria of a negative response.
- The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

OTHERS:
- Sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.

See attached document

Conclusions:
Under the test conditions, the test substance is not considered as mutagenic in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or in the absence of a rat liver metabolizing system.
Executive summary:

An in vitro gene mutation study in bacteria was performed according to the OECD Guideline 471 (bacteria reverse gene mutation assay) and in compliance with GLP.

A preliminary toxicity test was performed to define the dose-levels of the test substance dissolved in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.  Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).  Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48-72 hours of incubation at 37°C, the revertant colonies were scored.  The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. Since the test item was found freely soluble but toxic in the preliminary test mainly in the absence of S9 mix, the selection of the highest dose-level to be used in the main experiments was based on the level of toxicity.

Main experiments without S9 mix

Selected dose-levels ranged from 0.8 to 600 μg/plate.

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.

In the first experiment, only a slight toxicity (thinning of the bacterial lawn) was noted at the highest dose-level of 200 μg/plate in all the tested strains. The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains in this first experiment.

In the second experiment, a moderate to strong toxicity was noted at dose-levels ≥ 66.7 μg/plate in the TA 1535, TA 1537 and TA 100 strains, and ≥ 200 μg/plate in the TA 98 and TA 102 strains. These observations were consistent with the toxicity observed in the preliminary toxicity test. The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in this second experiment. These data were consistent with results of the first experiment performed under the same experimental conditions despite the use of a higher range of dose-levels.

The overall results without S9 mix met the criteria of a negative response.

Main experiments with S9 mix

Selected dose-levels ranged from 6.9 to 5000 μg/plate.

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.

In the first experiment, using the direct plate incorporation method, a moderate to strong toxicity was noted at dose-levels ≥ 2500 μg/plate in the TA 1535, TA 1537 and TA 100 strains, whereas no noteworthy toxicity was noted in the TA 98 and TA 102 strains.

In the second experiment, using the pre-incubation method, a moderate to strong toxicity was noted at dose-levels ≥ 312.5 μg/plate in the TA 1535, TA 1537 and TA 98 strains, ≥ 555.6 μg/plate in the TA 100 strain and ≥ 1250 μg/plate in the TA 102 strain.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment. These results with S9 mix met the criteria of a negative response.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

Under the test conditions, the test substance is not considered as mutagenic in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or in the absence of a rat liver metabolizing system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 February to 24 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD 476 Guideline without any deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
28 October 2016
Type of assay:
other: In vitro mammalian cell gene mutation test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TCH Consortium / 16/01806
- Appearance: Pale yellow liquid
- Expiration date of the lot/batch: 01 May 2018
- Purity test date: 15 November 2016

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4 °C, in the dark
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
- Cell cycle length, doubling time or proliferation index: The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type.
- Modal number of chromosomes: 22

MEDIA USED
- Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS))
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically checked for karyotype stability: Yes; cells have a stable karyotype
- Periodically 'cleansed' against high spontaneous background: Yes; Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM). Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix contains: S9 fraction (2%) prepared from male rats, dosed with phenobarbital and β-Naphthaflavone
Test concentrations with justification for top dose:
Preliminary cytotoxicity test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL, 4 h exposure without and with metabolic activation

Mutation tests:
4 h exposure with metabolic activation: 2.5, 5, 10, 15, 20, 25, 30, 35 and 40 μg/mL
4 h exposure with metabolic activation: 10, 20, 30, 40, 50, 60, 70 and 80 μg/mL

The maximum concentration selected for the main mutagenicity experiment was therefore limited by test item-induced toxicity in both the absence and presence of metabolic activation, as recommended by the OECD 476 guidelines.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Following solubility checks performed in-house for the Human Lymphocyte in vitro Micronucleus Test performed on the same test item (Envigo Study No. YV71HH), the test item was accurately weighed and formulated in acetone prior to serial dilutions being prepared. The test item was a UVCB, therefore, the maximum proposed concentration level in the solubility test was set at 5000 μg/mL initially, the maximum recommended concentration level, and no correction for the purity of the test item was applied. However, Acetone is toxic to V79 cells at dose volumes greater than 0. 5% of the total culture volume. Therefore, the test item was formulated at 500 mg/mL and dosed at 0.5% to give a maximum achievable concentration level of 2500 μg/mL in the subsequent preliminary toxicity test.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
Remarks:
with S9-mix
Details on test system and experimental conditions:
CELL CULTURE
The stock of cells was stored in liquid nitrogen. For use, a sample of cells were removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37 °C with 5% CO2 in humidified air.

METHOD OF APPLICATION: Eagles Minimal Essential (MEM) with 10% fetal bovine serum (FBS)
- Cell density at seeding: Cells were seeded at 1 x 10^7 cells/225 cm2 flask approximately 24 h being exposed to the test or control items.

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
All cell cultures were incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.

SELECTION AGENT (mutation assays): 6-Thioguanine (6-TG) at a final concentration of 11 μg/mL; At 2 x 10^5 cells/petri dish (ten replicates per group) in MEM with 10% FBS supplemented with 11 μg/mL 6-Thioguanine (6-TG), to determine mutant frequency.

NUMBER OF REPLICATIONS:
Preliminary toxicity test: Single culture for test item and vehicle controls
Main test: Duplicate cultures for test item, vehicle and positive controls

NUMBER OF CELLS EVALUATED:
200 cells/flask were seeded for cloning efficiency and 2 x 10^5 cells/flask were analyzed for mutant frequencies.

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency

- OTHER:
- Cytotoxicity flasks were incubated for 6 or 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted and recorded to estimate cytotoxicity.
- The percentage cloning efficiency and mutation frequency per survivor were calculated for each dose group.
- Fixation and staining of all flasks/petri dishes was achieved by aspirating off the media, washing with phosphate buffered saline, fixing for 5 minutes with methanol and finally staining with a 10% Giemsa solution for 5 minutes.
Rationale for test conditions:
The test item was a UVCB, therefore, the maximum proposed concentration level in the solubility test was set at 5000 μg/mL initially, which was the maximum recommended concentration level in regulatory guidelines.
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly positive if, in any of the experimental conditions examined:
i) At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) The increase is considered to be concentration-related.
iii) The results are outside the range of the historical negative control data for the test item concentrations.
When all these criteria are met, the test chemical is then considered able to induce gene mutations in cultured mammalian cells in this test system.
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in all of the experimental conditions examined:
i) None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
ii) There is no concentration related increase.
iii) The results for the test item concentrations are within the range of the historical negative control data.
The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
There is no requirement for verification of a clearly positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgment and/or further investigations. Performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, S9 concentration, and exposure time) may be useful.
Statistics:
When there is no indication of any increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
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: There was no significant change in pH when the test item was dosed into media at 9.77 to 2500 μg/mL.
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm at the concentration levels investigated (9.77 to 2500 μg/mL).
- Precipitation: At the end of the exposure period, precipitate of the test item was observed at and above 156.25 μg/mL.

PRELIMINARY CYTOTOXICITY TEST:
- A concentration range of 9.77 to 2500 μg/mL was used in the preliminary cytotoxicity test. The maximum concentration tested was the maximum achievable dose level.
- At the end of the exposure period, precipitate of the test item was observed at and above 156.25 μg/mL.
- There was evidence of marked concentration related reductions in cloning efficiency in both the absence and presence of metabolic activation.

MAIN TEST:
- At the end of the exposure period, the onset of precipitate of the test item was observed at 70 μg/ml in the absence of metabolic activation. Therefore, following the recommendations of the OECD 490 guideline, the 80 μg/mL dose level was excluded from the analysis as it was considered to be surplus to requirements.
- There were marked concentration-related reductions in the Day 0 cloning efficiency values in both the absence and presence of metabolic activation, and optimum levels of toxicity were considered to have been achieved. There was no evidence of any reductions in the Day 7 cloning efficiencies at any of the concentration levels, therefore indicating that residual toxicity had not occurred.
- The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.

HISTORICAL CONTROL DATA (mean and standard deviation)
- Positive historical control data:
Mutant Frequencies per survivor (x 10^-6): 226.52 ± 77.63 (Ethyl methanesulphonate, -S9); 319.07 ± 157.07 (Dimethyl benzanthracene, +S9)
- Negative (solvent/vehicle) historical control data:
Mutant Frequencies per survivor (x 10^-6): 13.92 ± 7.04 (-S9); 20.62 ± 13.64 (+S9)

None

Conclusions:
The test item did not induce any toxicologically significant or concentration-related increases in mutant frequency per survivor in either the absence or presence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.
Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, Chinese hamster (V79) cells were treated with the test item for 4 h, with and without metabolic activation (2% S9); S9 fraction prepared from male rats, dosed with phenobarbital and β-Naphthaflavone.

 

Preliminary cytotoxicity test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL, 4 h exposure without and with metabolic activation

 

Mutation tests:

4 h exposure without metabolic activation: 2.5, 5, 10, 15, 20, 25, 30, 35 and 40 μg/mL

4 h exposure with metabolic activation: 10, 20, 30, 40, 50, 60, 70 and 80 μg/mL

 

In a preliminary toxicity test, at the end of the exposure period, precipitate of the test item was observed at and above 156.25 μg/mL. There was evidence of marked concentration related reductions in cloning efficiency in both the absence and presence of metabolic activation. The maximum concentration selected for the main mutagenicity experiment was therefore limited by test item-induced toxicity in both the absence and presence of metabolic activation, as recommended by the OECD 476 guidelines.

 

In main test, at the end of the exposure period, the onset of precipitate of the test item was observed at 70 μg/ml in the absence of metabolic activation. Therefore, following the recommendations of the OECD 490 guideline, the 80 μg/mL dose level was excluded from the analysis as it was considered to be surplus to requirements. There were marked concentration-related reductions in the Day 0 cloning efficiency values in both the absence and presence of metabolic activation, and optimum levels of toxicity were considered to have been achieved. There was no evidence of any reductions in the Day 7 cloning efficiencies at any of the concentration levels, therefore indicating that residual toxicity had not occurred. The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.

 

The vehicle (acetone) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

 

The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 December 2016 to 15 March 2017
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted according to OECD 487 Guideline with deviation: only 284 binucleate cells analyzed for micronuclei at 80 μg/mL in the 4 h exposure group (-S9)
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
yes
Remarks:
only 284 binucleate cells analyzed for micronuclei at 80 μg/mL in the 4 h exposure group (-S9)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
28 October 2016
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: TCH Consortium / 16/01806
- Appearance: Pale yellow liquid
- Expiration date of the lot/batch: 01 May 2018
- Purity test date: 15 November 2016

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Approximately 4 °C in the dark
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
At the end of the exposure period in both experiments, the cell cultures were incubated for a further 24 h in the presence of Cytochalasin B at a final concentration of 4.5 μg/mL.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (2% final concentration of S9): S9 fraction, prepared from male rats dosed with phenobarbital and β-Naphthaflavone
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL; 4 h exposure with and without S9-mix; 24 h exposure without S9-mix

Main Test
4 h exposure to the test item formulations without S9-mix: 10, 20, 30, 40, 50, 60, 70 and 80 μg/mL
4 h exposure to the test item formulations with S9-mix: 20, 40, 50, 60, 70, 80, 100 and 120 μg/mL
24 h exposure to the test item without S9-mix: 5, 10, 20, 40, 50, 60, 70 and 80 μg/mL

Main Test - repeat
4 h exposure to the test item formulations without S9-mix: 30, 60, 80, 100, 120, 140 and 160 μg/mL
4 h exposure to the test item formulations with S9-mix: 60, 80, 120, 140, 160, 200, 280 and 320 μg/mL
24 h exposure to the test item without S9-mix: 7.5, 15, 30, 60, 80, 100, 120 and 160 μg/mL

The test item could not be formulated at 1000 mg/mL, therefore, the maximum practical concentration was limited to 2500 μg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- The test item was considered to be a UVCB and therefore the maximum recommended dose was initially set at 5000 μg/mL. Additionally, the test item was considered to be 100% so no correction for purity made.
The test item was immiscible in dimethyl sulphoxide at 500 mg/mL but was fully miscible in acetone at the same concentration in solubility checks performed in-house. Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed in 50 μL aliquots. The test item could not be formulated at 1000 mg/mL, therefore, the maximum practical concentration was limited to 2500 μg/mL.
Prior to each experiment, the test item was accurately weighed, formulated in acetone and serial dilutions prepared.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
other: Demecolcine
Remarks:
without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Details on test system and experimental conditions:
CELLS:
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 h.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 25 years; Main Experiment: male, aged 25 years; Main Experiment Repeat male, aged 33 years

CULTURE OF LYMPHOCYTES:
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 °C with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
Culture conditions:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture: 9.05 mL MEM, 10% (FBS); 0.1 mL Li-heparin; 0.1 mL phytohaemagglutinin; 0.75 mL heparinized whole blood

METHOD OF APPLICATION: in medium; Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS).

DURATION
- Exposure duration: 4 h (± S9) and 24 h exposure (-S9) in preliminary toxicity test; 4 h (± S9) and 24 h exposure (-S9) in main experiments
- Fixation time: 24 h incubation period in treatment-free media after exposure to the test item in all experiments

SPINDLE INHIBITOR (cytogenetic assays): At the end of the exposure period in both experiments, the cell cultures were washed and then incubated for a further 24 h in the presence of Cytochalasin B at a final concentration of 4.5 μg/mL.

STAIN (for cytogenetic assays): Slides were stained in 5% Giemsa for 5 minutes

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture was prepared for test item and vehicle control
- Main tests: Duplicate cultures were prepared for test item, vehicle and positive controls

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Cell harvest: At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.
Preparation of microscope slides: The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Then the slides were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED:
- A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls.
- The micronucleus frequency in 2000 binucleated cells was analyzed per concentration (1000 binucleated cells per culture, two cultures per concentration). Due to excessive test item toxicity, only 284 binucleate cells could be analyzed for micronuclei in the B culture at 80 μg/mL in the 4 h exposure group without S9-mix. Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis Block Proliferation Index (CBPI)
The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B. It was used to calculate cytostasis by the following formula:
% Cytostasis = 100 - 100{(CBPIT – 1) / (CBPIC – 1)}
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total n
umber of cells]
T = test item treatment culture
C = vehicle control culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for identifying micronuclei were that they were round or oval in shape, non-refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.

- OTHER:
Qualitative slide assessment: The slides were checked microscopically to determine the quality of the binucleate cells and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for CBPI evaluation.
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. The Study Director may make a judgement based on experience and the biological relevance of the data and any justification for acceptance of the data will be included in the report.
Statistics:
The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.
Key result
Species / strain:
lymphocytes: human
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: There was no significant change in pH when the test item was dosed into media at 9.77 to 2500 μg/mL
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm at 9.77 to 2500 μg/mL when compared to vehicle control.
- Precipitation: Yes

PRELIMINARY TOXICITY TEST
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 312.5 μg/mL in all three exposure groups.
- Hemolysis was observed following exposure to the test item at all the dose levels tested in the 4 h exposure groups whereas hemolysis was observed at and above 19.53 μg/mL in the 24 h continuous exposure group. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. In addition to hemolysis, a reduced pellet was observed at and above 78.13 μg/mL in the 4 h exposure groups whereas a reduced pellet was observed at and above 156.25 μg/mL in the 24 h continuous exposure group. A reduced pellet is an indication of cytotoxicity and that maximum exposure is occurring. This type of toxic response to the lymphocytes is not reflected in the CBPI data because the surviving cells are still dividing.
- Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 39.06 μg/mL in the exposure groups in the absence of metabolic activation (S9). The maximum dose with binucleate cells present in the presence of S9 was 78.13 μg/mL. The test item induced evidence of severe toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment was, therefore, based on toxicity for all three exposure groups.

MAIN EXPERIMENT
- In the Main Experiment, the qualitative assessment of the slides determined that the toxicity was less than that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the maximum dose level of test item in all three exposure groups which did not achieve optimum toxicity as defined in the OECD 487 guideline (55±5%). No precipitate of test item was noted at any of the dose levels in any exposure groups tested.
- Hemolysis was observed following exposure to the test item at all the dose levels tested in the 4 h exposure groups whereas hemolysis was observed at and above 20 μg/mL in the 24 h continuous exposure group. Also and in addition to the hemolysis, a reduced pellet was observed at and above 60 and 70 μg/mL in the 4 h exposure groups in the absence and presence of S9, respectively, whereas no reduced pellet was observed at any of the dose levels in the 24 h continuous exposure group. Therefore, the hemolysis and reduced pellet were similar to that observed in the preliminary toxicity test.
- The CBPI data for the short exposure group in the presence of S9 only and for the 24 h exposure group confirm the qualitative observations in that no or only a moderate dose-related inhibition of CBPI was observed. In the presence of S9, only a moderate inhibition of cell proliferation was observed where only 24% cytostasis was achieved at the maximum dose level (120 μg/mL). In the absence of S9, a dose-related inhibition of CBPI was observed in the 24 h continuous exposure group where 17%, 33% and 43% cytostasis was achieved at 60, 70 and 80 μg/mL, respectively. In the 4 h group, only a moderate inhibition of CBPI was observed. Therefore, the experiment was repeated in an effort to achieve optimum toxicity in all three exposure groups.

MAIN EXPERIMENT – REPEAT
- In the Main Experiment Repeat, the qualitative assessment of the slides determined that the toxicity was more than that observed in the original Main Experiment and more like the observations made in the Preliminary toxicity test.
- No precipitate of test item was noted at any of the dose levels in the 4 h exposure group in the absence of S9 but precipitate was observed at and above 280 μg/mL in the presence of S9 and at 160 μg/mL in the 24 h exposure group.
- Hemolysis was observed following exposure to the test item at all the dose levels tested in the 4 h exposure groups whereas hemolysis was observed at and above 60 μg/mL in the 24 h continuous exposure group. Also and in addition to the hemolysis, a reduced pellet was observed at and above 60 and 80 μg/mL in the 4 h exposure groups in the absence and presence of S9, respectively, whereas a reduced pellet was observed at 160 μg/mL only in the 24 h continuous exposure group. The cytotoxicity to lymphocytes that resulted in reduced cell pellets with little effect on the CBPI data was used as a justification for selecting maximum dose levels for analysis in some exposure groups.
- The qualitative assessment of the 4 h exposure group in the presence of S9 confirms that there were only two dose levels where there was enough binucleates for analysis present. In the 24 h continuous exposure group, very steep toxicity was observed where 10% and 74% cytostasis was observed at 80 and 100 μg/mL, respectively. Therefore, and with consultation with the Sponsor, the exposure groups and dose levels selected for micronuclei analysis were selected over the experiments as follows:
4 h exposure group in the absence of S9 (Main Experiment Repeat): 0, 30, 60 and 80 μg/mL
4 h exposure group in the presence of S9 (Main Experiment): 0, 60, 80 and 100 μg/mL
24 h continuous exposure group (Main Experiment): 0, 50, 60, 70 and 80 μg/mL
- The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
% binucleate cells with micronuclei: 2.25-18.85 (6.31 ± 2.90) - Mitomycin C (4 h, -S9); 1.75-5.75 (3.73 ± 1.06) - Demecolcine (24 h, -S9); 1.65-7.25 (3.48 ± 1.51) - Cyclophosphamide (4 h, +S9)
- Negative (solvent/vehicle) historical control data:
% binucleate cells with micronuclei:
4 h, -S9: 0.10-1.25 (0.38 ± 0.27); 95% CI - ± 0.09 (0.29-0.47)
24 h, -S9: 0.05-0.90 (0.43 ± 0.20); 95% CI - ± 0.07 (0.36-0.50)
4 h, +S9: 0.10-1.20 (0.41 ± 0.24); 95% CI - ± 0.08 (0.33-0.49)

Table 7.6.1/1: CBPI and Micronucleus Data – Main Experiment

Exposure Time +/- S9

Dose Level (μg/mL)

Replicate

Nucleate cells /500 cells

CBPI

% Cytostasis

Micronuclei (MN) per 1000 Binucleate cells

% Binucleate cells with MN

Mean % Binucleate cells with MN

Mono

Bi

Multi

1 MN

2     MN

>2 MN

4 h Exposure Without Metabolic Activation (-S9)

4 h (-S9)

0

A

136

283

81

1.89

0

5

0

0

0.50

0.60

B

104

311

85

1.96

7

0

0

0.70

30

A

167

267

66

1.80

10

1

0

0

0.10

0.45

B

127

305

68

1.88

7

1

0

0.80

60

A

262

221

17

1.51

47

8

0

0

0.80

0.75

B

277

209

14

1.47

7

0

0

0.70

80

A

328

157

15

1.37

59

2

0

0

0.20

1.33

B#

322

165

13

1.38

5

2

0

2.46

Mitomycin C  0.2

A

234

248

18

1.57

35

38

0

0

3.80

4.15***

B

199

291

10

1.62

43

2

0

4.50

4 h Exposure With Metabolic Activation (+S9)

4 h (+S9)

0

A

258

215

27

1.54

0

3

0

1

0.40

0.30

B

292

191

17

1.45

2

0

0

0.20

60

A

321

164

15

1.39

20

6

1

0

0.70

0.60

B

316

169

15

1.40

4

1

0

0.50

80

A

355

130

15

1.32

24

4

0

0

0.40

0.40

B

305

173

22

1.43

4

0

0

0.40

100

A

353

132

15

1.32

26

6

1

0

0.70

0.50

B

313

168

19

1.41

3

0

0

0.30

Cyclophosphamide 5

A

361

135

4

1.29

48

52

9

5

6.60

6.25***

B

390

108

2

1.22

49

7

3

5.90

24 h Exposure Without Metabolic Activation (-S9)

24 h (-S9)

0

A

139

343

18

1.76

0

1

0

0

0.10

0.30

B

149

334

17

1.74

5

0

0

0.50

50

A

165

322

13

1.70

9

5

0

0

0.50

0.40

B

181

310

9

1.66

3

0

0

0.30

60

A

192

298

10

1.64

17

9

0

0

0.90

0.60

B

210

287

3

1.59

3

0

0

0.30

70

A

239

259

2

1.53

33

3

0

1

0.40

0.50

B

277

214

9

1.46

5

0

1

0.60

80

A

251

247

2

1.50

43

4

0

0

0.40

0.25

B

329

169

2

1.35

1

0

0

0.10

Demecolcine 0.075

A

276

199

25

1.50

31

21

3

0

2.40

2.25***

B

253

228

19

1.53

13

7

1

2.10

*** = P<0.001

# Only 284 binucleates cells available for analysis

Conclusions:
Under the experimental conditions, the test item did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to the test item in the presence and absence of a metabolic activation system. Metabolic activation system used in this test was 2% S9; S9 fraction was obtained from the liver homogenates of male rats induced with phenobarbital and β-Naphthaflavone.

 

Preliminary Toxicity Test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL; 4 h exposure with and without S9-mix; 24 h exposure without S9-mix

Main Test

4 h exposure to the test item formulations without S9-mix: 10, 20, 30, 40, 50, 60, 70 and 80 μg/mL

4 h exposure to the test item formulations with S9-mix: 20, 40, 50, 60, 70, 80, 100 and 120 μg/mL

24 h exposure to the test item without S9-mix: 5, 10, 20, 40, 50, 60, 70 and 80 μg/mL

Main Test - repeat

4 h exposure to the test item formulations without S9-mix: 30, 60, 80, 100, 120, 140 and 160 μg/mL

4 h exposure to the test item formulations with S9-mix: 60, 80, 120, 140, 160, 200, 280 and 320 μg/mL

24 h exposure to the test item without S9-mix: 7.5, 15, 30, 60, 80, 100, 120 and 160 μg/mL

Cytokinesis was blocked following mitosis using Cytochalasin B. Then the cells were harvested and slides prepared, so that binucleate cells could be examined for micronucleus induction.

All vehicle (acetone) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Based on the toxicity, the dose levels selected for micronuclei analysis were as follows:

4 h exposure group in the absence of S9 (Main Experiment Repeat): 0, 30, 60 and 80 μg/mL

4 h exposure group in the presence of S9 (Main Experiment): 0, 60, 80 and 100 μg/mL

24 h continuous exposure group (Main Experiment): 0, 50, 60, 70 and 80 μg/mL

 

The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

 

It was concluded that the test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test:

An in vitro gene mutation study in bacteria was performed according to the OECD Guideline 471 (bacteria reverse gene mutation assay) and in compliance with GLP.

A preliminary toxicity test was performed to define the dose-levels of the test substance dissolved in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.  Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).  Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48-72 hours of incubation at 37°C, the revertant colonies were scored.  The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. Since the test item was found freely soluble but toxic in the preliminary test mainly in the absence of S9 mix, the selection of the highest dose-level to be used in the main experiments was based on the level of toxicity.

Main experiments without S9 mix

Selected dose-levels ranged from 0.8 to 600 μg/plate.

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.

In the first experiment, only a slight toxicity (thinning of the bacterial lawn) was noted at the highest dose-level of 200 μg/plate in all the tested strains. The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains in this first experiment.

In the second experiment, a moderate to strong toxicity was noted at dose-levels ≥ 66.7 μg/plate in the TA 1535, TA 1537 and TA 100 strains, and ≥ 200 μg/plate in the TA 98 and TA 102 strains. These observations were consistent with the toxicity observed in the preliminary toxicity test. The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in this second experiment. These data were consistent with results of the first experiment performed under the same experimental conditions despite the use of a higher range of dose-levels.

The overall results without S9 mix met the criteria of a negative response.

Main experiments with S9 mix

Selected dose-levels ranged from 6.9 to 5000 μg/plate.

No precipitate was observed in the Petri plates when scoring the revertants at any of the tested dose-levels.

In the first experiment, using the direct plate incorporation method, a moderate to strong toxicity was noted at dose-levels ≥ 2500 μg/plate in the TA 1535, TA 1537 and TA 100 strains, whereas no noteworthy toxicity was noted in the TA 98 and TA 102 strains.

In the second experiment, using the pre-incubation method, a moderate to strong toxicity was noted at dose-levels ≥ 312.5 μg/plate in the TA 1535, TA 1537 and TA 98 strains, ≥ 555.6 μg/plate in the TA 100 strain and ≥ 1250 μg/plate in the TA 102 strain.

The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment. These results with S9 mix met the criteria of a negative response.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were five analysable dose-levels for each strain and test condition. The study was therefore considered to be valid.

Under the test conditions, the test substance is not considered as mutagenic in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or in the absence of a rat liver metabolizing system.

Micronucleus test in human lymphocytes:

In anin vitromicronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to the test item in the presence and absence of a metabolic activation system. Metabolic activation system used in this test was 2% S9; S9 fraction was obtained from the liver homogenates of male rats induced with phenobarbital andβ-Naphthaflavone.

Preliminary Toxicity Test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL; 4 h exposure with and without S9-mix; 24 h exposure without S9-mix

Main Test

4 h exposure to the test item formulations without S9-mix: 10, 20, 30, 40, 50, 60, 70 and 80 μg/mL

4 h exposure to the test item formulations with S9-mix: 20, 40, 50, 60, 70, 80, 100 and 120 μg/mL

24 h exposure to the test item without S9-mix: 5, 10, 20, 40, 50, 60, 70 and 80 μg/mL

Main Test - repeat

4 h exposure to the test item formulations without S9-mix: 30, 60, 80, 100, 120, 140 and 160 μg/mL

4 h exposure to the test item formulations with S9-mix: 60, 80, 120, 140, 160, 200, 280 and 320 μg/mL

24 h exposure to the test item without S9-mix: 7.5, 15, 30, 60, 80, 100, 120 and 160 μg/mL

All vehicle (acetone) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

 It was concluded that the test item was considered to be non-clastogenic and non-aneugenic to human lymphocytesin vitro.

HPRT test in V79 cells:

In anin vitromammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP,Chinese hamster (V79) cells were treated with the test itemfor 4 h, with and without metabolic activation (2% S9); S9 fraction prepared from male rats, dosed with phenobarbital and β-Naphthaflavone.

 Preliminary cytotoxicity test: 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL, 4 h exposure without and with metabolic activation

 Mutation tests:

4 h exposure without metabolic activation: 2.5, 5, 10, 15, 20, 25, 30, 35 and 40 μg/mL

4 h exposure with metabolic activation: 10, 20, 30, 40, 50, 60, 70 and 80 μg/mL

 In apreliminary toxicity test, at the end of the exposure period, precipitate of the test item was observed at and above 156.25 μg/mL. 

In main test, at the end of the exposure period, the onset of precipitate of the test item was observed at 70 μg/mL in the absence of metabolic activation. Therefore, following the recommendations of the OECD 490 guideline, the 80 μg/mL dose level was excluded from the analysis as it was considered to be surplus to requirements. There were marked concentration-related reductions in the Day 0 cloning efficiency values in both the absence and presence of metabolic activation, and optimum levels of toxicity were considered to have been achieved. There was no evidence of any reductions in the Day 7 cloning efficiencies at any of the concentration levels, therefore indicating that residual toxicity had not occurred. The test item did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test, in either the absence or presence of metabolic activation.

 The vehicle (acetone) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

 The test item was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available information, no classification is proposed according to the Regulation (EC) No. 1272/2008 and GHS.