Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The objective was the evaluation of the genetic toxicity of the test substance, by in vitro test models.

All three tests were negative.

OECD guideline 471

The test substance was tested in the Bacterial Reverse Mutation Test (Ames test) according to OECD guideline 471. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies of Salmonella typhimuriumand in the number of revertant (Trp+) colonies of Escherichia coli, both in the absence and presence of S9-metabolic activation. 

Based on the results of this study it is concluded that the test item is not mutagenic in both the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay.

OECD guideline 473

The test substance was tested in the Mammalian Chromosome Aberration Test, according to OECD Guideline 473.

Both in the absence and presence of S9-mix the test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations; this was observed in two independent experiments.

Therefore it is concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the applied experimental conditions. 

OECD guideline 490:

In a GLP-compliant OECD guideline 490 study, the test item was not mutagenic in the mouse lymphoma test both with and without metabolic activation in two independent experiments with 3 and 24 hours exposure, when tested up to cytotoxic or precipitating concentrations.

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:
27 April 2017 - 18 May 2017
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
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium strains TA1535, TA1537, TA98, TA100: histidine gene
Escherichia coli WP2uvrA strain: tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
0, 17, 52, 164, 512, 1600 and 5000 µg/plate (+/- S9 mix)
The dose-range finding test was performed with the strains TA100 and the WP2uvrA, both with and without S9-mix, using the following concentrations: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Vehicle / solvent:
Ethanol. The stock solution was completely dissolved after sonication. Test item concentrations were used within 2 hours after preparation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Negative solvent / vehicle controls:
yes
Remarks:
Saline
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
For TA1535 strain, without metabolic activation, at concentration of 5 µg, in both direct plate assay and pre-incubation assay
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: ICR-191 (Sigma)
Remarks:
For TA1537 strain, without metabolic activation, at concentration of 2.5 µg (used only in direct plate assay, not in pre-incubation assay)
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
2-nitrofluorene
Remarks:
For TA1537 strain, without metabolic activ., at conc. of 15 µg, only in the preincubation assay and not in Direct plate assay. For TA98 strain, without metabolic activ., at conc. 10 micrograms, in both direct plate assay and in preincubation assay
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
For TA100 strain, without metabolic activation, at concentration of 650 µg, in both direct plate assay and pre-incubation assay.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
For WP2uvrA strain, without metaboilic activation, at concentration of 10 µg, in both direct plate assay and pre-incubation assay
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
other: 2-aminoanthracene (Sigma)
Remarks:
For TA1535, TA1537, TA98, TA100, WP2uvrA, with metabolic activ, in both direct plate and preincubation assay. Conc: TA1535 and TA1537= 2.5 µg; TA98 = 1 µg, TA100 = 1 µg in direct plate and 5 µg in preincubation ; WP2uvrA = 15 µg
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
2-nitrofluorene
Remarks:
For TA98 strain, without metabolic activation, at concentration of 10 µg in both direct plate assay and preincubation assay
Details on test system and experimental conditions:
METHOD OF APPLICATION:
in agar (plate incorporation); test concentrations were prepared immediately before use.

NUMBER OF REPLICATIONS:
triplicate

DETERMINATION OF CYTOTOXICITY
bacterial background lawn

EXPOSURE DURATION
48 +/- 4 h
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The vehicle control and positive control plates from each tester strain (with or without
S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
At the end of the incubation period, the test item precipitated from the concentration of 512 µg/plate in the absence of S9-mix, from 1600 µg/plate in the presence of S9-mix, and heavily at 5000 µg/plate with S9 mix.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Since the test item heavily precipitated at concentration of 5000 µg/plate without S9-mix, the cytotoxicity at this dose could not be determined
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
At the end of the incubation period, the test item precipitated from the concentration of 512 µg/plate in the absence of S9-mix, from 1600 µg/plate in the presence of S9-mix, and heavily at 5000 µg/plate with S9 mix.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Since the test item precipitated heavily at the concentration of 5000 microgr/plate without S9-mix, at this dose level cytotoxicity could not be determined
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
At the end of the incubation period, the test item precipitated from the concentration of 512 µg/plate in the absence of S9-mix, from 1600 µg/plate in the presence of S9-mix, and heavily at 5000 µg/plate with S9 mix.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Since the test item heavily precipitated at concentration of 5000 µg/plate, cytotoxicity at this concentration could not be determined
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
At the end of the incubation period, the test item precipitated from the concentration of 1600 µg/plate in the absence of S9-mix, and heavily at 5000 µg/plate with S9 mix.
Cytotoxicity / choice of top concentrations:
other: Cytotoxicity was not observed in presence of S9-mix; cytotoxicity was observed in absence of S9-mix. Since the test item precipitated heavily at concentration of 5000 µg/plate in presence of S9-mix, cytotoxicity at this dose could not be determined
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
At the end of the incubation period, the test item heavily precipitated at the concentration of 5000 µg/plate in presence and absence of S9 mix.
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
No cytotoxicity was observed up to the dose level of 1600 µg/plate. Since the test item heavily precipitated at the concentration of 5000 µg/plate in presence and absence of S9 mix, cytotoxicity could not be determined at this dose level
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Test item precipitated at the start of the incubation period at the concentration of 5000 µg/plate and at 1600 and 5000 µg/plate at the end of the incubation period
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Since the test item heavily precipitated at the concentration of 5000 µg/plate in presence and absence of S9 mix, cytotoxicity could not be determined at this dose level.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Test item precipitated at the start of the incubation period at the concentration of 5000 µg/plate, and at 1600 and 5000 µg/plate at the end of the incubation period
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Since the test item heavily precipitated at the concentration of 5000 µg/plate in presence and absence of S9 mix, cytotoxicity could not be determined at this dose level.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Test item precipitated at the start of the incubation period at the concentration of 5000 µg/plate, and at 1600 and 5000 µg/plate at the end of the incubation period
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Since the test item heavily precipitated at the concentration of 5000 µg/plate in presence and absence of S9 mix, cytotoxicity could not be determined at this dose level.
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Precipitation at start of incubation at 5000 µg/plate, at end incubation at 1600 and 5000 µg/plate. At 17 and 512 µg/plate with S9-mix up to 4fold increases vs solv control, due to low solv control values. Increases within historical control data range.
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Since the test item heavily precipitated at the concentration of 5000 µg/plate in presence and absence of S9 mix, cytotoxicity could not be determined at this dose level
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Test item precipitated at the start of the incubation period at the concentration of 5000 µg/plate, and at 1600 and 5000 µg/plate at the end of the incubation period
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Since the test item heavily precipitated at the concentration of 5000 µg/plate in presence and absence of S9 mix, cytotoxicity could not be determined at this dose level.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
DOSE RANGE finding study was performed with the strains TA100 and WP2 uvrA, both with and without S9-mix, at the concentration of: 1.7; 5.4; 17; 52; 164; 512; 1600; 5000"µg/plate . Those were tested in triplicate. The results are shown in the "test results" table, above, under teh remark "direct plate assay".

Remarks on result:
other: This result from Pre-incubation assay
Conclusions:
Interpretation of results (migrated information):
negative.

In an Ames test, the test substance showed to be negative with and without metabolic activation in 5 strains: TA1535, TA1537, TA98, TA100 and WP2 uvrA
Executive summary:

The test substance was tested in the Bacterial Reverse Mutation Test with histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100), and with a tryptohan-requiring strain of Escherichia coli (WP2uvrA) in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

The study was performed according to OECD guideline 471. Six concentrations (17, 52, 164, 512, 1600 and 5000 µg/plate) were tested in triplicate, each assay was conducted twice (direct mutagenicity plate test and pre-incubation mutation test).

In the direct mutagenicity plate test, the test item precipitated on the plates at dose levels of 1600 and 5000 μg/plate. In the pre-incubation mutation experiment, the test item precipitated with all the strains at dose levels of 5000 µg/plate in presence of the metabolic activation system, and in the plates containing WP2uvrA at dose levels of 5000 µg/plate in absence of metabolic activation system.

In the plates with the precipitate, neither the bacterial background lawn nor the number of revertants of this dose level could be determined.

In the other concentration ranges, the test item did not induce a significant dose-related increase in the number of revertant (His+) colonies of Salmonella typhimuriumand in the number of revertant (Trp+) colonies of Escherichia coli, both in the absence and presence of S9-metabolic activation. 

Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 26 April 2017 to 07 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
other: In Vitro Mammalian Chromosome Aberration Test
Target gene:
Human lymphocytes, either in the presence and in the absence of a metabolic activation system (S9-mix)
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
cultured human lymphocytes
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Dose-range finding test:
9.8, 20, 39, 78, 156, 313 µg test item/ml culture medium with and without S9-mix (1.8% v/v), with 24 h exposure time and 24 h fixation time, and with 48 h exposure time and 48 h fixation time. The test item precipitated in the culture medium at the concentrations of 156 and 313 µg /ml culture

First cytogenetic assay:
39, 78 and 156 µg test item/ml culture medium with and without S9-mix with 3 h exposure time and 24 h fixation time. The test item precipitated in the culture medium at the concentration of 156 /ml culture

Second cytogenetic assay:
0.5, 5, 10, 20, 30, 40, 50 µg test item/ml culture medium without S9-mix, with 24 h exposure time and 24 h fixation time.
0.5, 5, 10, 15, 20, 25, 30 µg test item/ml culture medium without S9-mix, with 48 h exposure time and 48 h fixation time.

Based on the observations from the above concentrations, the following doses were selected for scoring of chromosome aberration:
0.5, 20, 30 µg test item/ml culture medium without S9-mix, with 24 h exposure time and 24 h fixation time.
0.5, 5, 15 µg test item/ml culture medium without S9-mix, with 48 h exposure time and 48 h fixation time.
Vehicle / solvent:
Ethanol
In the dose range finding study/first cytogenetic assay, the test item was heated (80°C) for 25 minutes, after which the stock solution was treated with ultrasonic waves until the test item had completely dissolved. In the second cytogenetic assay the test item was dissolved by treatment with ultrasonic waves.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Negative solvent / vehicle controls:
yes
Remarks:
Hanks’ Balanced Salt Solution (HBSS) (Life technologies, Bleiswijk, The Netherlands), without calcium and magnesium
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
mitomycin C
Remarks:
Used as a direct acting mutagen at a final concentration of 0.5 and 0.75 µg/ml for a 3 h exposure period, 0.2 and 0.3 µg/ml for a 24 h exposure period and 0.1 and 0.15 µg/ml for a 48 h exposure period
Negative solvent / vehicle controls:
yes
Remarks:
Hanks’ Balanced Salt Solution (HBSS) (Life technologies, Bleiswijk, The Netherlands), without calcium and magnesium.
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
cyclophosphamide
Remarks:
used as an indirect acting mutagen, requiring metabolic activation, at a final concentration of 10 µg/ml for a 3 h exposure period.
Details on test system and experimental conditions:
TEST SYSTEM
Cultured peripheral human lymphocytes were used as test system.
Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to
35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2016)

LYMPHOCYTES CULTURES
Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Remel, Europe Ltd., Dartford, United Kingdom) was added

ENVIRONMENTAL CONDITIONS
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 38 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.8 - 37.2°C).

EXPERIMENTAL DESIGN
Lymphocytes (0.4 ml blood of a healthy donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 48 ± 2 h and thereafter exposed to selected doses of the test item
The cytogenetic assay was carried out as described by Evans, 1984 (2) with minor modifications
The lymphocytes were cultured in duplicate

FURTHER INFORMATION ON THE FIRST CYTOGENETIC ASSAY
After 3 h exposure to the test item in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 ml culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h and 48 h fixation time).
Cytotoxicity of the test item in the lymphocyte cultures was determined using the mitotic index.

ACTIVATION SYSTEM
The test item was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate.

CHROMOSOME PREPARATION
During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/ml medium) (Acros Organics, Geel, Belgium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride (Merck) solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck): acetic acid (Merck) fixative (3:1 v/v).

PREPARATION OF THE SLIDES
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for
10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

MITOTIC INDEX / DOSE SELECTION FOR SCORING OF THE CYTOGENETIC ASSAY
The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%). At least three analyzable concentrations were used for scoring of the cytogenetic assay. In the first cytogenetic assay, the test substance was not cytotoxic and difficult to dissolve in aqueous solutions, the highest concentration analyzed was determined by the solubility in the culture medium. For the second cytogenetic assay, chromosomes of metaphase spreads were analyzed from those cultures with an inhibition of the mitotic index of 55 ± 5%, whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations

ANALYSIS OF SLIDES FOR CHROMOSOME ABERRATION
To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with Charles River Den Bosch study identification number and code was placed over the marked slide. One hundred and fifty metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 38 in 75 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analyzed. The number of cells with aberrations and the number of aberrations were calculated. Since the lowest concentration of MMC-C resulted in a positive response the highest concentration was not examined for chromosome aberrations
Evaluation criteria:
A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analyzed by the Fisher’s exact test (one-sided, p < 0.05).
Key result
Species / strain:
lymphocytes: Cultured Human Lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
The possible clastogenicity of the test item was tested in two independent experiments
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Determined using the mitotic index
Vehicle controls validity:
valid
Remarks:
Ethanol
Untreated negative controls validity:
valid
Remarks:
Ethanol
Positive controls validity:
valid
Remarks:
Mitomycin C used with metabolic activation, Cyclophosphamide used without metabolic activation
Additional information on results:
In the dose-range finding test, the test item precipitated in the culture medium at the concentrations of 156 and 313 µg /ml culture

The lymphocytes were cultured in duplicate at all the concentrations of the test item

In the second cytogenetic assay, the following doses were selected for scoring of chromosome aberrations:
Without S9-mix:
- 0.5, 20 and 30 µg/ml culture medium (24 h exposure time, 24 h fixation time).
- 0.5, 5 and 15 µg/ml culture medium (48 h exposure time, 48 h fixation time).
Remarks on result:
other:
Remarks:
The test item did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments. No biologically relevant effects of the test item on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Temporary deviations from the temparture, humidity and CO2 percentage may occur due to the opening and closing of the incubator door. Based on laboratory historical data, these deviations are considered not to affect the study integrity

Conclusions:
The performed test is valid and the test item is not clastogenic in human lymphocytes under the applied experimental conditions.
Executive summary:

The test substance was tested according to OECD Guideline 473 (in vitro Mammalian Chromosome Aberration Test).

The ability of the test substance to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments.

In the first cytogenetic assay, the test substance was tested up to 156 µg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. The test substance precipitated in the culture medium at the highest dose level. 

In the second cytogenetic assay, the test substance was tested up to 30 µg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 15 µg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals (mitomycin C and cyclophosphamide) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Both in the absence and presence of S9-mix the test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No biologically relevant effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the applied experimental conditions. 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 February 2018 - 6 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable

OTHER SPECIFICS: no correction for purity was applied.
Target gene:
Thymidine kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001)
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD).

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2mM L-glutamin. Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (R10-medium). Exposure medium: basic medium supplemented with 5% (v/v) (3-hours exposure) or 10% (24 hours exposure) heat-inactivated horse serum (R5- and R-10 media, respectively). Selective medium: basic medium, supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium) and 5 μg/mL trifluorothymidine (TFT). Non-selective medium: basic medium, supplemented with 20% (v/v) heat-inactivated horse serum (R20-medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
S9 homogenate from phenobarbital and beta-naphthoflavone induced male Sprague-Dawley rats
Test concentrations with justification for top dose:
Dose-range finding test, 3 hours exposure:
-S9: 0, 9.4, 19, 38, 75, 150 and 300 μg/mL (the top dose chosen based on the solubility in the exposure medium)
+S9: 0, 8.5, 17, 35, 68, 136 and 273 μg/mL
Dose-range finding test, 24 hours exposure:
-S9: 0, 9.4, 19, 38, 75, 150 and 300 μg/mL
Experiment 1, 3 hours exposure:
- S9: 0, 2.5, 5, 10, 20, 40, 60, 80, 90, 100, 110, 130, 140 and 150 μg/mL (doses selected for measuring mutation frequencies: 2.5, 5, 10, 20, 40, 60, 80 and 90 μg/mL)
+ S9: 0, 6.3, 12.5, 25, 50, 70, 90, 100, 110, 120, 130, 140 and 150 μg/mL (doses selected for measuring mutation frequencies: 6.3, 25, 70, 100, 110, 120, 130 and 140 μg/mL)
Experiment 2, 24 hours exposure:
-S9: 0, 5, 15, 25, 35, 45, 55, 65, 75 and 85 μg/mL (doses selected for measuring mutation frequencies: 5, 15, 25, 35, 45, 55, 65 and 75 μg/mL).


Vehicle / solvent:
Vehicle(s)/solvent(s) used: ethanol (maximal concentration 1% in the medium)
- Justification for choice of solvent/vehicle: recommended by the guideline
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 9.6 x 10^5 cells/concentration, 2000 cells/well for the treatment groups and sovent controls, 1000 cells/well for positive controls

DURATION
- Exposure duration: 3 or 24 hours (experiments 1 and 2, respectively)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT), 5 μg/mL

NUMBER OF REPLICATIONS: 2 independent experiments run in parallel; 5 plates/concentration, solvent controls tested in duplicate

STAINING TECHNIQUE USED: staining with 0.5 mg/mL MMT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) for 2 hours

NUMBER OF CELLS EVALUATED: 2000 cells/well for the treatment groups and sovent controls, 1000 cells/well for positive controls

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


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
- Any supplementary information relevant to cytotoxicity:
For determination of the cloning efficiency 2 days after the treatment period (CEday2) the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
The Relative Total Growth (RTG) was calculated as the product of the cumulative relative suspension growth (RSG) and the relative survival for each culture.
Evaluation criteria:
Evaluation criteria:
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. Any observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
Statistics:
A Cochran Armitage trend test (p < 0.05) was performed to test whether there is a significant trend in the induction of mutation frequency (ToxRat Professional v 3.2.1).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
The test substance precipitated directly in the exposure medium at concentrations of 75 μg/mL and above. After 3 hours, the test item precipitated in the exposure medium at concentrations of 150 μg/mL and above.
In experiment 1, the test item precipirated in the exposure medium at concentrations of 130 and 140 μg/mL with metabolic activation. The test item was tested beyond the limit of the solubility to obtain adequate mutagenicity data.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see section "Any other information on results including tables"
- Negative (solvent/vehicle) historical control data: see section "Any other information on results including tables"

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In a dose-range finding test with 3 an 24 hours exposure, the relative suspension growth in the absence of S9 mix following 3 hours exposure was 37% at the test item concentration of 75 μg/mL compared to the relative suspension growth of the solvent control. No or hardly any cell survival was observed at test item concentrations of 150 μg/mL and above. In the presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the test item concentration of 68 μg/mL compared to the relative suspension growth of the solvent control. No or hardly any cell survival was observed at test item concentrations of 136 μg/mL and above.
Following 24 hours exposure, the relative suspension growth was 16% at the test item concentration of 75 μg/mL compared to the relative suspension growth of the solvent control. No or hardly any cell survival was observed at test item concentrations of 150 μg/mL and above.

In experiment 1, the dose levels of 100 to 150 μg/mL were not used for mutation frequency measurement, since these dose levels were too toxic for further testing or showed an inconsistent RSG (110 µg/mL). In the presence of S9-mix, the dose levels of 6.3 to 100 μg/mL showed no cytotoxicity. Therefore, the dose levels of 12.5, 50 and 90 µg/mL were not regarded relevant for mutation frequency measurement.
In experiment 1, relative total growth (RTG) was reduced to at the highest test item concentration of 90 μg/mL to 4% in the absence of S9-mix and to 2% at the highest test item concentration of 140 μg/mL in the presence of S9 mix.

In experiment 2, the dose level of 85 μg/mL was not used for mutation frequency measurement, since this dose level was too toxic for further testing. The RTG was reduced to 9% at the highest test concentratin of 75 μg/mL.

ADDITIONAL INFORMATION ON ACCEPTABILITY OF THE ASSAY:
The mutation frequency found in the solvent control cultures was within the range of the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical concurrent solvent control database, except in the second experiment in which the mutation frequency of the solvent control cultures was not within the range of the acceptability criteria. Since the mutation frequencies were just above the upper limit of the acceptability criteria range and clear negative results are observed in all experiments, this deviation in the mutation frequency had no effect on the validity of the results of the second mutation experiment.

Historical Control Data of the Spontaneous Mutation Frequencies of the Solvent Controls for the Mouse Lymphoma Assay

 

Mutation frequency per 106survivors

 

- S9-mix

+ S9-mix

 

3 hour treatment

24 hour treatment

3 hour treatment

Mean

96

92

96

SD

29

30

29

n

268

248

285

Upper control limit

(95% control limits)

160

152

160

Lower control limit

(95% control limits)

32

31

32

SD = Standard deviation

n = Number of observations

Distribution historical negative control data from experiments performed between November 2014 and November 2017. 

Historical Control Data of the Mutation Frequencies of the Positive Controls for the Mouse Lymphoma Assay

 

Mutation frequency per 106survivors

 

- S9-mix

+ S9-mix

 

3 hour treatment

24 hour treatment

3 hour treatment

Mean

808

684

1669

SD

239

206

843

n

136

124

146

Upper control limit

(95% control limits)

1465

1222

4196

Lower control limit

(95% control limits)

152

146

-859

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between November 2014 and November 2017. 

Conclusions:
In a GLP-compliant OECD guideline 490 study, the test item was not mutagenic in the mouse lymphoma test both with and without metabolic activation when tested up to either cytotoxic or precipitating concentrations.
Executive summary:

In a GLP-compliant OECD guideline 490 study the ability of the test substance to increase mutation frequency in mammalian cells in vitro was tested in a mouse lymphoma assay in L5178Y cell line. Two independent experiments were performed, one with 3 hours exposure both with and without metabolic activation, and another with 24 hours exposure without metabolic activation. The test item was dissolved in ethanol. In the first experiment, the test substance was evaluated up to and including concentrations of 90 and 140 µg/mL in the absence and presence S9-mix, respectively. Relative total growth (RTG) was reduced to 4 and 2% at the highest evaluated concentrations in the absence and presence of S9-mix, respectively. The test substance precipitated in the culture medium at the dose levels of 130 and 140 µg/mL. In the second experiment, the test substance was tested up to concentrations of 75 µg/mL in the absence of S9-mix. The RTG was reduced to 9% at the highest tested concentration. The test substance did not precipitate in the culture medium at this dose level. The test substance did not induce a significant increase in the mutation frequency in any experiment both with and without metabolic activation. The positive controls produced satisfactory response. Based on this the assay is considered to be valid, and it can be concluded that test substance is not mutagenic under the condition of this assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

The substance is not genotoxic.

Additional information

OECD guideline 471

The test substance was tested in the Bacterial Reverse Mutation Test with histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100), and with a tryptophan-requiring strain of Escherichia coli (WP2uvrA) in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

The study was performed according to OECD guideline 471, using six concentrations, tested in triplicate. Each assay was conducted twice (direct mutagenicity plate test and pre-incubation mutation test).

In the direct mutagenicity plate test, the test item precipitated on the plates at dose levels of 1600 and 5000 μg/plate. In the pre-incubation mutation experiment, the test item precipitated with all the strains at dose levels of 5000 µg/plate in presence of the metabolic activation system, and in the plates containing WP2uvrA at dose levels of 5000 µg/plate in absence of metabolic activation system.

In the plates with the precipitate, neither the bacterial background lawn nor the number of revertants of this dose level could be determined.

In the other concentration ranges,the test item did not induce a significant dose-related increase in the number of revertant (His+) colonies of Salmonella typhimurium and in the number of revertant (Trp+) colonies of Escherichia coli, both in the absence and presence of S9-metabolic activation. 

OECD guideline 473

The test substance was tested according to OECD Guideline 473 (in vitro Mammalian Chromosome Aberration Test).

The ability of the test substance to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments. Those were performed with different test substance concentrations, different exposure times and different fixation times.

Appropriate toxicity was reached.

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals (mitomycin C and cyclophosphamide) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Both in the absence and presence of S9-mix the test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No biologically relevant effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. 

OECD guideline 490:

In a GLP-compliant OECD guideline 490 study the ability of the test substance to increase mutation frequency in mammalian cells in vitro was tested in a mouse lymphoma assay inL5178Y cell line. Two independent experiments were performed, one with 3 hours exposure both with and without metabolic activation, and another with 24 hours exposure without metabolic activation. The test item was dissolved in ethanol.In the first experiment,

the test substance was evaluated up to and including concentrations of 90 and 140 µg/mL in the absence and presence S9-mix, respectively. Relative total growth (RTG) was reduced to 4 and 2% at the highest evaluated concentrations in the absence and presence of S9-mix, respectively. The test substance precipitated in the culture medium at the dose levels of 130 and 140 µg/mL. In the second experiment, the test substance was tested up to concentrations of 75 µg/mL in the absence of S9-mix. The RTG was reduced to 9% at the highest tested concentration. The test substance did not precipitate in the culture medium at this dose level. The test substance did not induce a significant increase in the mutation frequency in any experiment both with and without metabolic activation. The positive controls produced satisfactory response, and the acceptability criteria were met. Based on this the assay is considered to be valid, and it can be concluded that the test substance is not mutagenic under the condition of this assay.

Justification for classification or non-classification

OECD guideline 471

In the Ames test, the test item did not induce a significant dose-related increase in the number of revertant (His+) colonies of Salmonella typhimurium and in the number of revertant (Trp+) colonies of Escherichia coli, both in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

OECD guideline 473

The test substance was tested in the Mammalian Chromosome Aberration Test, according to OECD Guideline 473.

Both in the absence and presence of S9-mix the test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations; this was observed in two independent experiments.

No biologically relevant effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. 

Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the applied experimental conditions. 

OECD guideline 490:

In a GLP-compliant OECD guideline 490 study, the test item was not mutagenic in the mouse lymphoma test both with and without metabolic activation in two independent experiments with 3 and 24 hours exposure, when tested up to cytotoxic or precipitating concentrations.

Conclusions

The test substance was tested according to the following in vitro tests: Bacterial Reverse Mutation Test, Mammalian Chromosome Aberration Test and Mammalian Gene Mutation (Mouse Lymphoma) Test.

Since all three in vitro test were negative, no in vivo mutagenicity study is needed, according to REACH Annex VIII, 8.4, column 2.

Based on these results, the test substance does not have to be classified and has no obligatory labelling requirement for mutagenic toxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).