Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not induce gene mutations or chromosomal aberrations in the V79 Chinese hamster cell line nor gene mutations in S. typhimurium bacterial cells in the presence and absence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 March 1986 to 25 April 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1983
Deviations:
not specified
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
1982
Deviations:
not specified
GLP compliance:
yes
Type of assay:
in vitro mammalian cell transformation assay
Target gene:
Chinese hamster cell line V79
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Laboratory stock
- Suitability of cells: The V79 cell line has been used for many years in in vitro experiments with success. The high proliferation rate and plating efficiency of untreated cells are necessary for the appropriate performance of the study.
- Cell cycle length, doubling time or proliferation index: Doubling time 12 to 16 hours in stock cultures.
- Methods for maintenance in cell culture: Thawed stock cultures were propagated at 37°C in plastic flasks. Seeding was done with 100000 to 300000 cells per flask in 5 mL of MEM-medium supplemented with 10% fetal calf serum. Cells were subcultured twice a week.
- Modal number of chromosomes: 22
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Test concentrations with justification for top dose:
The highest concentration should reduce the mitotic index to about 50 % as compared to the solvent control.
7 hour preparation time: 10 μg/mL (without S9) and 36 μg/mL (with S9)
18 hour preparation time: 1, 5 and 11 μg/mL (without S9) and 3, 15 and 36 μg/mL (with S9)
28 hour preparation time: 11 μg/mL (without S9) and 36 μg/mL (with S9)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (1%)
- Justification for choice of solvent/vehicle: Ethanol is relatively non-toxic in the concentrations applied.
Untreated negative controls:
yes
Remarks:
untreated cultures
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethyl methanesulfonate (without metabolic activation), cyclophosphamide (with metabolic activation)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in suspension
- Cell density at seeding: Two days old logarithmically growing stock cultures more than 50% confluent were trypsinised and a single cell suspension was prepared. The cells were seeded into four parallel plastic tissue culture flasks at a concentration of 300000 cells per flask (7 hour interval) and 150000 cells per flask (18 and 28 hour intervals).

DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 5, 15.5 and 25.5 hours after the start of treatment
- Fixation time: 2.0 (7 hour interval) or 2.5 (18 and 28 hour intervals) hours from addition of colcemid

STAIN: Aceto-orcein

NUMBER OF REPLICATIONS: 4

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were trypsinised and suspended in a hypotonic solution (37.5 mM KCl) for 10 minutes at 37°C. After centrifugation (800 to 1000 rpm) the cell pellets were fixed in three steps with glacial acetic acid/ methanol 1 + 3 and stored overnight at 4°C. The next day the cells were spread on wet and ice cold slides and stained.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 100 metaphases/flask were scored per test concentration.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index
- Any supplementary information relevant to cytotoxicity: Mitotic index was determined in 1000 cells in each replicate.
Rationale for test conditions:
The test item concentrations were determined in a preliminary experiment using the mitotic index and plating efficiencies as an indicator for toxicity response.
Evaluation criteria:
Gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural aberrations.
1. The test substance is classified as mutagenic if it induces with one of the concentrations tested a significantly increased aberration rate as compared with the negative control. The significance is obvious either by an enhancement of the rate clearly over the control range or it is proven by adequate biometry.
2. The test substance is classified as mutagenic if there is a concentration related increase in the aberration rates as compared with the solvent controls.
3. The test substance is considered negative when 1 and 2 do not apply.
Statistics:
No statistical analysis performed as there is no indication of a dose-response relationship.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: See Table 2.

The sensitivity of the test system was demonstrated by the clearly enhanced structural aberration rates found in the groups treated with the positive control substances.

In only one group of cells treated with the lowest concentration of the test item, a higher number of cells with structural chromosome aberrations as compared to the range of the negative controls was found in the presence of S9 mix at preparation interval 18 h. In all other groups treated with the test item, aberration rates within or near the control range were found. This single higher value alone is not regarded as being sufficient to prove mutagenicity.

Table 1. Mutagenicity data and mitotic index

 Preparation time  Treatment group  Total cells analysed  Number of aberrant cells including gaps  % of aberrant cells including gaps  Number of aberrant cells excluding gaps  % of aberrant cells excluding gaps  % of aberrant cell exchanges  % mitotic index (absolute)  % mitotic index (relative)
 18 hours  Untreated control -S9  400  9  2.25  5  1.25  0.25  6.8  100.0
 18 hours  Untreated control +S9  400  24  6.00  7  1.75  0.00  6.0  100.0
 18 hours  Solvent control -S9  400  17  4.25  4  1.00  0.00  4.8  100.0
 18 hours  Solvent control +S9  400  14  3.50  9  2.25  0.25  5.4  100.0
 18 hours  Positive control -S9  200  60  60.00  52  52.00  45.00  7.7  113.2
 18 hours  Positive control +S9  200  69  69.00  65  65.00  39.00  4.3  71.6
 18 hours  1 μg/mL test item -S9  400  23  5.75  10  2.50  0.00  4.6  95.6
 18 hours  3 μg/mL test item +S9  400  37  9.25  19  4.75  0.00  4.5  83.6
 18 hours  5 μg/mL test item -S9  400  19  4.75  5  1.25  0.25  5.6  115.6
 18 hours  15 μg/mL test item +S9  400  13  3.25  4  1.00  0.50  2.9  54.2
 18 hours  11 μg/mL test item -S9  400  17  4.25  11  2.75  0.50  3.2  65.8
 18 hours  36 μg/mL test item +S9  400  21  5.25  5  1.25  0.00  7.5  139.8
 7 hours  Solvent control -S9  400  16  4.00  4  1.00  0.00  5.9  100.0
 7 hours  Solvent control +S9  400  10  2.50  1  0.25  0.00  5.9  100.0
 7 hours  10 μg/mL test item -S9 400   14  3.50  8  2.00  0.00  2.8  47.7
 7 hours  36 μg/mL test item +S9  400  28  7.00  13  3.25  0.00  5.2  86.9
 28 hours  Solvent control -S9  400  17  4.25

 6

 1.50  0.25  4.4  100.0
 28 hours  Solvent control +S9  400  10  2.50  2  0.50  0.00  1.9  43.1
 28 hours  11 μg/mL test item -S9  400  11 2.75   6  1.50  0.5  5.7  100.0
 28 hours  36 μg/mL test item +S9  400  25  6.25  10  2.50  0.00  5.7  99.1

Table 2. Preliminary study results

 Treatment group  Number of seeded cells per flask Mean number of found cells per flask  % PE (absolute)  % PE (relative)
 Untreated control -S9  496  300.0  60.5  100.0
 Untreated control +S9  498  435.0  87.3  100.0
 Solvent control -S9  496  311.5  62.8  100.0
 Solvent control +S9  498  387.0  77.7  100.0
 10.0 μg/mL test item -S9  496  224.0  45.2  71.9
 11.0 μg/mL test item -S9  496  155.5  31.4  49.9
 12.0 μg/mL test item -S9  496  20.0  4.0  6.4
 13.0 μg/mL test item -S9  496  17.0  3.4  5.5
 14.0 μg/mL test item -S9  496  0.0  0.0  0.0
 15.0 μg/mL test item -S9  496  0.0  0.0  0.0
 15.0 μg/mL test item +S9  498  356.5  71.6  92.1
 20.0 μg/mL test item +S9  498  377.5  75.8  97.5
 25.0 μg/mL test item +S9  498  350.5  70.4  90.6
 30.0 μg/mL test item +S9  498  342.0  68.7  88.4
 35.0 μg/mL test item +S9  498  233.5  46.9  60.3
 40.0 μg/mL test item +S9  498  2.0  0.4  0.5
Conclusions:
The test item was not mutagenic to V79 Chinese hamster cell line in vitro in the presence and absence of metabolic activation.
Executive summary:

The potential of the test item to induce chromosomal aberrations was determined in vitro with cells of the V79 Chinese hamster cell line according to OECD Guideline 473. Chinese hamster lung fibroblasts V79 were treated with the test item at concentrations of 1, 5, 10 and 11 µg/mL without S9-activation and 3, 15 and 36 µg/mL with S9-activation. Cells were incubated with the test item for 4 hours, followed by a 5, 15.5 or 25.5 -hour incubation period in growth medium. Colcemid was added as a fixing agent and the cells were incubated for a further 2.0 or 2.5 hours. The study included negative, solvent and positive (ethyl methanesulfonate and cyclophosphamide) controls . Mutagenicity was evaluated by determining the number of gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations in 400 metaphases per test concentration. Cytotoxicity was evaluated by determination of mitotic index in 1000 cells in each replicate. The test item was not mutagenic to V79 Chinese hamster cell line in the presence and absence of metabolic activation. This study is considered reliable without restriction (Klimisch 1).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not applicable
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
and TA 1538
Additional strain / cell type characteristics:
other: histidine mutation, uvrB deletion, rfa deletion, R-factor presence
Metabolic activation:
with and without
Metabolic activation system:
Liver postmitochondrial fraction (S-9 from male rats induced with Aroclor 1254)
Test concentrations with justification for top dose:
100, 500, 1000, 5000, 10000 and 15000 μg/plate
Toxic effect sought in the highest concentration.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
methylmethanesulfonate
other: 2-aminoanthracene, daunomycin, 1-ethyl-2-nitro-3-nitrosoguanidine, N-methyl-N-nitro-N-nitrosoguanidine; 4-nitro-o-phenylene diamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 8 for the solvent control, 4 for the test item

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Colonies were stained with triphenyltetrazolium if counting of bacteria colonies is affected by precipitation.

OTHER:
- Plate composition: 0.1 mL bacteria suspension, 0.1 mL test solution, 0.5 mL Na-phosphate buffer (in trials without metabolic activation), 0.5 mL S-9 mix (in trials with metabolic activation), 2.0 mL soft agar solution
Statistics:
Standard deviations were calculated.
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:
not examined
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:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
not examined
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:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: Toxic effect was not achieved at the highest dose due to low solubility of the test substance in the test medium.
- Precipitation: The compound precipitated from the test medium at the dose levels of 5000, 10000 and 15000 μg/plate.
- Definition of acceptable cells for analysis: Where counting of bacterial colonies was not possible because of precipitation of the test substance, the colonies were stained with triphenyltetrazolim chloride before evaluation of the plates. This staining enables the colonies to be distinguished from the precipitate and can then be counted as usual.

HISTORICAL CONTROL DATA
- Positive historical control data: Results were acceptable and within the normal range
- Negative (solvent/vehicle) historical control data: Results were acceptable and within the normal range.

Table 1. First trial for mutagenic effects without metabolic activation

 Test item concentration (μg/plate)  Revertant colonies per plate (± standard deviation)
   Base-pair substitution        Frame-shift   
   TA 100  TA 1535  TA 98  TA 1537  TA 1538
 Solvent control (ethanol)  62 ± 8  10 ± 3  31 ± 7  5 ± 1  8 ± 3
 100  57 ± 6 10 ± 3  28 ± 8  5 ± 1  7 ± 4
 500  65 ± 10  7 ± 3  30 ± 6  6 ± 1  9 ± 5
 1000  44 ± 9  8 ± 2  27 ± 7  5 ± 1  7 ± 2
 5000  50 ± 8  5 ± 1  30 ± 9  3 ± 1  8 ± 3
 10000  73 ± 13  6 ± 0  34 ± 6  4 ± 1  6 ±1
 15000  75 ± 10  5 ± 4  36 ± 2  5 ± 0  7 ± 2

Table 2. First trial for mutagenic effects with metabolic activation

 Test item concentration (μg/plate)  Revertant colonies per plate (± standard deviation)
   Base-pair substitution        Frame-shift   
   TA 100  TA 1535  TA 98  TA 1537  TA 1538
 Solvent control  74± 10  13± 6  52± 9  9± 3  20± 4
 100  55± 15 11±3  39± 5  7± 4  17± 3
 500  70± 15  9± 2  41± 8  7± 3  14± 4
 1000  71± 9  10± 3  50± 13  7± 2  14± 6
 5000  61± 5  11± 0  48± 11  8± 3  12± 2
 10000  68± 3  12± 3  50± 2  7± 2  18± 6
 15000  76± 6  14± 5  42± 8  6± 1  18± 5

Table 3. Second (repeat) trial for mutagenic effects without metabolic activation

 Test item concentration (μg/plate)  Revertant colonies per plate (± standard deviation)
   Base-pair substitution        Frame-shift   
   TA 100  TA 1535  TA 98  TA 1537  TA 1538
 Solvent control  96±10  11± 2  22± 7  7± 2  6± 1
 100  87± 13 6±2  25± 3  6± 3  5± 2
 500  106± 26  9± 4  20± 3  5 ± 2  5± 1
 1000  106± 17  12± 5  21± 3  4± 3  3± 2
 5000  97± 5  4± 2  31± 4  5± 1  4 ± 2
 10000  107± 8  5± 4  9± 1  2± 2

 2± 1

 15000

 106± 12

 9± 1

 18± 5

 3± 2

 5± 1

Table 4. Second (repeat) trial for mutagenic effects with metabolic activation

 Test item concentration (μg/plate)  Revertant colonies per plate (± standard deviation)
   Base-pair substitution        Frame-shift   
   TA 100  TA 1535  TA 98  TA 1537  TA 1538
 Solvent control  97±17  16± 5  30± 8  5± 2  17± 2
 100  119± 17 15±3  24± 3  4± 2  13± 2
 500  112± 15  11± 1  40± 17  7± 3  10± 1
 1000  128± 12  12± 2  24± 10  3 ± 1  15± 5
 5000  125± 5  17± 5  25± 7  3± 2  11± 5
 10000  124± 15  8± 3  41± 1  6± 4

 11± 4

 15000

 122± 11

 10± 3

 28± 4

 3± 1

 5± 2

Conclusions:
The test substance did not show mutagenic activity in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 up to 15000 μg/plate, with and without metabolic activation.
Executive summary:

The mutagenic potential of the test item was determined in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 using a plate incorporation method similar to OECD Guideline 471. The test substance was incubated with and without metabolic activation (rat liver S-9 mix) at nominal concentrations of 100, 500, 1000, 5000, 10000 and 15000 μg/plate. Two experiments were performed, each consisting of 4 test item replicates and 8 solvent control (ethanol) replicates. 2 -aminoanthracene, 9 -aminoacridine, daunomycin, 1 -ethyl-2 -nitro-3 -nitrosoguanidine, methyl methanesulfonate, N-methyl-N-nitro-N-nitrosoguanidine, 2 -nitrofluorene and 4 -nitro-1,2 -phenylenediamine were tested as the positive controls. The test substance did not show mutagenic activity in any S. typhimurium strain up to 15000 μg/plate, with and without metabolic activation. This study is reliable without restrictions (Klimisch 1).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 July 2017 to 24 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Kanpoan No. 287 (Environmental Protection Agency), Eisei No. 127 (Ministry of Health & Welfare), Heisei 09/10/31 Kikyoku No. 2 (Ministry of International Trade & Industry)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian cell gene mutation test using the Hprt and xprt genes (migrated information)
Target gene:
Hprt and xprt
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Laboratory cell bank.
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years, particularly due to the doubling time and cloning efficiency.
- Cell cycle length, doubling time or proliferation index: 12-16 h doubling time in stock cultures and more than 50% cloning efficiency of untreated cells.
- Methods for maintenance in cell culture if applicable: Storage in liquid nitrogen.
- Modal number of chromosomes: 22

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells were cultured in MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%). During treatment no FBS was added to the medium. All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
With metabolic activation: 0.75, 1.5, 3, 6, 8, 10, 12, 16 and 20 μg/mL
Without metabolic activation: 0.78, 1.6, 3.1, 6.25, 12.5, 25, 50 and 100 μg/mL
The top dose was chosen to have a relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO (1.0% v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen for its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 2.0×10^6 cells per test vessel

DURATION
- Preincubation period: 24 hours
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 8 days

STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: 5 (+ 2 for determination of cytotoxicity)

NUMBER OF CELLS EVALUATED: Stained colonies with more than 50 cells were counted.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
- Any supplementary information relevant to cytotoxicity: Cloning efficiency was determined after exposure to the test item (survival) and after the expression time (viability).
Rationale for test conditions:
Two parallel cultures were used throughout the assay. In order to establish a concentration response effect of the test item at least four concentration levels are tested. To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.
Evaluation criteria:
A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).

A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).

In cases when the response is neither clearly negative nor clearly positive as described above, or in order to judge the biological relevance of a result, the data was evaluated by expert judgement or further investigations.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A trend is judged as significant whenever the p-value (probability value) is below 0.05 when compared to the solvent control groups. A t-Test was performed to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05. Both biological and statistical significance were considered together.
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 examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant shift of pH of the medium even at the maximum concentration of the test item.
- Effects of osmolality: No relevant shift of osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: Precipitation was observed at the end of the treatment in the three highest test concentation without metabolic activation (12, 16 and 20 μg/mL) and the two highest test concentration with metabolic activation (50 and 100 μg/mL). Mutation rate was not assessed for these precipitating test concentrations (except at 50 μg/mL, mutation rate was assessed).
- Other confounding effects: Severe cytotoxicity was observed in the four highest test concentation without metabolic activation (10, 12, 16 and 20 μg/mL), therefore mutation rate was not assessed for these cytotoxic test concentrations. The data at 8.0 μg/mL without metabolic activation were rejected as severe cytotoxicity was observed, therefore the mutant frequency was not taken into the consideration of the evaluation of this assay.

RANGE-FINDING/SCREENING STUDIES: The first pre-experiment was performed in the presence and absence of metabolic activation for test item concentrations between 15.6 and 2000 μg/mL. A relevant cytotoxic effect (indicated by a relative cloning efficiency of 50% or below) was observed at 15.6 μg/mL and above without metabolic activation and at 125.0 μg/mL and above with metabolic activation. Due to strong toxic effects in the absence of S9 mix, this part of the pre-experiment was repeated with lower concentrations (0.78 μg/mL to 50 μg/mL). In the second pre-experiment, relevant cytotoxic effect was observed at 12.5 μg/mL and above without metabolic activation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Number of mutant colonies per 10^6 cells, without metabolic activation: mean 190.3, range 53.9 to 872.3, standard deviation 88.4, 95% confidence limit not reported. Number of mutant colonies per 10^6 cells, with metabolic activation: mean 215.8, range 56.7 to 739.9, standard deviation 110.9, 95% confidence limit not reported.
- Negative (solvent) historical control data: Number of mutant colonies per 10^6 cells, without metabolic activation: mean 15.9, range 3.4 to 41.0, standard deviation 7.1, 95% confidence limit 1.7 to 30.2. Number of mutant colonies per 10^6 cells, with metabolic activation: mean 15.7, range 2.4 to 39.2, standard deviation 6.8, 95% confidence limit 2.0 to 29.4.

Table 1. Summary of results for culture 1

 Treatment

 S9 mix

 Relative cloning efficiency (%)

 Relative cell density (%)

 Relative adjusted cloning efficiency (%)  Number of mutant colonies / 10^6 cells  95% confidence interval
Solvent control   -  100.0  100.0  100.0  21.8  1.7 - 30.2
 Positive control (300.00 μg/mL EMS)  -  103.5  85.1  88.1  253.0  1.7 - 30.2
 Test item (0.75 μg/mL)  -  101.7  108.3  110.1  14.8  1.7 - 30.2
 Test item (1.50 μg/mL)  -  102.1  91.0  92.9  10.5  1.7 - 30.2
 Test item (3.00 μg/mL)  -  102.4  106.4  109.0  21.4  1.7 - 30.2
 Test item (6.00 μg/mL)  -  102.7  89.5  91.9  30.3  1.7 - 30.2
 Test item (8.00 μg/mL) (X)  -  10.4  53.6  5.6  43.9  1.7 - 30.2
 Test item (10.00 μg/mL)  -  7.0  28.0  2.0  -  1.7 - 30.2
 Test item (12.00 μg/mL) (P)  -  -  2.9  ##  ##  1.7 - 30.2
 Test item (16.00 μg/mL) (P)  -  ##  ##  ##  ##  1.7 - 30.2
 Test item (20.00 μg/mL) (P)  -  ##  ##  ##  ##  1.7 - 30.2
 Solvent control  +  100.0  100.0  100.0  7.2  2.0 - 29.4
 Positive control (2.30 μg/mL DMBA)  +  81.0  98.0  79.4  124.3  2.0 - 29.4
 Test item (0.78 μg/mL)  +  -  109.6  #  -  2.0 - 29.4
 Test item (1.60 μg/mL)  +  94.1  124.2  116.9  -  2.0 - 29.4
 Test item (3.10 μg/mL)  +  84.4  121.0  102.1  5.9  2.0 - 29.4
 Test item (6.25 μg/mL)  +  93.1  119.8  111.5  14.6  2.0 - 29.4
 Test item (12.50 μg/mL)  +  90.9  114.9  104.4  15.3  2.0 - 29.4
 Test item (25.00 μg/mL)  +  88.0  114.7  100.9  28.6  2.0 - 29.4
 Test item (50.00 μg/mL) (P)  +  75.0  98.4  73.8  15.8  2.0 - 29.4
 Test item (100.00 μg/mL) (P)  +  -  8.1  ##  ##  2.0 - 29.4

EMS: Ethylmethane sulfonate

DMBA: 7,12 -dimethylbenz(a)anthracene

(P): Precipitation visible at the end of treatment

(X): Data not considered in evaluation due to severe cytotoxicity

#: Culture was not continued as a minimum of only four analysable concentrations are required

##: Culture was not continued due to severe cytotoxic effects

-: Not reported

Table 2. Summary of results for culture 2

 Treatment

 S9 mix

 Relative cloning efficiency (%)

 Relative cell density (%)

 Relative adjusted cloning efficiency (%)  Number of mutant colonies / 10^6 cells  95% confidence interval
Solvent control   -  100.0  100.0  100.0  10.0  1.7 - 30.2
 Positive control (300.00 μg/mL EMS)  -  98.3  71.1  69.8  234.3  1.7 - 30.2
 Test item (0.75 μg/mL)  -  98.3  88.1  86.6  23.3  1.7 - 30.2
 Test item (1.50 μg/mL)  -  104.0  62.4  64.8  15.6  1.7 - 30.2
 Test item (3.00 μg/mL)  -  101.4  77.2  78.3  12.8  1.7 - 30.2
 Test item (6.00 μg/mL)  -  100.7  82.2  83.4  11.0  1.7 - 30.2
 Test item (8.00 μg/mL) (X)  -  11.4  42.4  4.8  41.3  1.7 - 30.2
 Test item (10.00 μg/mL)  -  2.7  28.6  0.8  -  1.7 - 30.2
 Test item (12.00 μg/mL) (P)  -  -  1.1  ##  ##  1.7 - 30.2
 Test item (16.00 μg/mL) (P)  -  ##  ##  ##  ##  1.7 - 30.2
 Test item (20.00 μg/mL) (P)  -  ##  ##  ##  ##  1.7 - 30.2
 Solvent control  +  100.0  100.0  100.0  7.4  2.0 - 29.4
 Positive control (2.30 μg/mL DMBA)  +  81.0  71.6  58.0  149.5  2.0 - 29.4
 Test item (0.78 μg/mL)  +  #  #  #  #  2.0 - 29.4
 Test item (1.60 μg/mL)  +  94.8  58.1  55.1  -  2.0 - 29.4
 Test item (3.10 μg/mL)  +  99.3  69.2  68.7  16.0  2.0 - 29.4
 Test item (6.25 μg/mL)  +  94.4  66.1  62.5  21.7  2.0 - 29.4
 Test item (12.50 μg/mL)  +  49.5  70.0  34.6  24.4  2.0 - 29.4
 Test item (25.00 μg/mL)  +  55.4  84.5  46.9  14.6  2.0 - 29.4
 Test item (50.00 μg/mL) (P)  +  39.7  45.9  18.3  20.9  2.0 - 29.4
 Test item (100.00 μg/mL) (P)  +  -  8.1  ##  ##  2.0 - 29.4

EMS: Ethylmethane sulfonate

DMBA: 7,12 -dimethylbenz(a)anthracene

(P): Precipitation visible at the end of treatment

(X): Data not considered in evaluation due to severe cytotoxicity

#: Culture was not continued as a minimum of only four analysable concentrations are required

##: Culture was not continued due to severe cytotoxic effects

-: Not reported

Conclusions:
The test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test item is considered to be non-mutagenic in the HPRT assay.
Executive summary:

The potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was determined according to OECD Guideline 476, EU Method B.17, EPA OPPTS 870.5300 and Japanese guidelines. The study was conducted in medium at test item concentrations of 0.75, 1.5, 3, 6, 8, 10, 12, 16 and 20 μg/mL in the absence metabolic activation and 0.78, 1.6, 3.1, 6.25, 12.5, 25, 50 and 100 μg/mL in the presence of metabolic activation. A solvent control and positive controls were tested concurrently. Five replicates were assessed per test item treatment and control, and two parallel cultures were used throughout the assay. The treatment period was 4 hours with and without metabolic activation, with a 24 hour pre-exposure period and 7 day expression (post-exposure) period in medium. Cells were fixed and stained with 10% methylene blue in 0.01% KOH solution 8 days after treatment. Precipitation and / or severe cytotoxicity were observed in the four highest test concentation without metabolic activation (10, 12, 16 and 20 μg/mL) and at the highest test concentration with metabolic activation (100 μg/mL), therefore mutation frequency was not assessed for these concentrations. Severe cytotoxicity also occurred in the 8 μg/mL treatment level without metabolic activation, and this data was not taken into the consideration of the evaluation of this assay. No relevant and reproducible increase in mutant colony numbers per 10^6 cells was observed with and without metabolic activation. Therefore, the test item is considered to be non-mutagenic in the HPRT assay. This study is considered to be reliable without restrictions (Klimisch 1).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Three in vitro studies are available to determine the genetic toxicity of the test item.

The mutagenic potential of the test item to bacteria was determined in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 using a plate incorporation method similar to OECD Guideline 471 (1980). The test substance was incubated with and without metabolic activation (rat liver S-9 mix) at nominal concentrations of 100, 500, 1000, 5000, 10000 and 15000 μg/plate. Two experiments were performed, each consisting of 4 test item replicates and 8 solvent control (ethanol) replicates. 2 -aminoanthracene, 9 -aminoacridine, daunomycin, 1 -ethyl-2 -nitro-3 -nitrosoguanidine, methyl methanesulfonate, N-methyl-N-nitro-N-nitrosoguanidine, 2 -nitrofluorene and 4 -nitro-1,2 -phenylenediamine were tested as the positive controls. The test substance did not show mutagenic activity in any S. typhimurium strain up to 15000 μg/plate, with and without metabolic activation. This study is reliable without restrictions (Klimisch 1).

The potential of the test item to induce chromosomal aberrations was determined in vitro with cells of the V79 Chinese hamster cell line according to OECD Guideline 473 (1986). Chinese hamster lung fibroblasts V79 were treated with the test item at concentrations of 1, 5, 10 and 11 µg/mL without S9-activation and 3, 15 and 36 µg/mL with S9-activation. Cells were incubated with the test item for 4 hours, followed by a 5, 15.5 or 25.5 -hour incubation period in growth medium. Colcemid was added as a fixing agent and the cells were incubated for a further 2.0 or 2.5 hours. The study included negative, solvent and positive (ethyl methanosulfonate and cyclophosphamide) controls. Mutagenicity was evaluated by determining the number of gaps, breaks, fragments, deletions, exchanges and chromosomal disintegrations in 400 metaphases per test concentration. Cytotoxicity was evaluated by determination of mitotic index in 1000 cells in each replicate. The test item was not mutagenic to V79 Chinese hamster cell line in the presence and absence of metabolic activation. This study is considered reliable without restrictions (Klimisch 1).

The potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster was determined according to OECD Guideline 476, EU Method B.17, EPA OPPTS 870.5300 and Japanese guidelines (2017). The study was conducted in medium at test item concentrations of 0.75, 1.5, 3, 6, 8, 10, 12, 16 and 20 μg/mL in the absence metabolic activation and 0.78, 1.6, 3.1, 6.25, 12.5, 25, 50 and 100 μg/mL in the presence of metabolic activation. A solvent control and positive controls were tested concurrently. Five replicates were assessed per test item treatment and control, and two parallel cultures were used throughout the assay. The treatment period was 4 hours with and without metabolic activation, with a 24 hour pre-exposure period and 7 day expression (post-exposure) period in medium. Cells were fixed and stained with 10% methylene blue in 0.01% KOH solution 8 days after treatment. Precipitation and / or severe cytotoxicity were observed in the four highest test concentation without metabolic activation (10, 12, 16 and 20 μg/mL) and at the highest test concentration with metabolic activation (100 μg/mL), therefore mutation frequency was not assessed for these concentrations. Severe cytotoxicity also occurred in the 8 μg/mL treatment level without metabolic activation, and this data was not taken into the consideration of the evaluation of this assay. No relevant and reproducible increase in mutant colony numbers per 10^6 cells was observed with and without metabolic activation. Therefore, the test item is considered to be non-mutagenic in the HPRT assay. This study is considered to be reliable without restrictions (Klimisch 1).

Justification for classification or non-classification

Three in vitro studies are available to evaluate the genotoxicity of the test item. All studies were considered reliable without restriction (Klimisch 1) and sufficient for classification. None of studies indicated any potential for mutagenicity, clastogenicity or cytogenicity. Consequently, in accordance with the CLP Regulation (EC) No. 1272/2008, the test item is not classified as a genetic toxicant.