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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance was not mutagenic/genotoxic in three in vitro GLP guideline studies:

- Ames test in bacteria (Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA; OECD TG 471);

- Micronucleus test in human lymphocytes (the substance was non-clastogenic and non-aneugenic) (OECD TG 487);

- HPRT test in hamster V79 lung fibroblasts (OECD TG 476).

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:
14 July 2017 to 22 September 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
The bacteria used in this assay did not possess the mammalian enzyme system that is known to convert promutagens into mutagenic metabolites.
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
The bacteria used in this assay did not possess the mammalian enzyme system that is known to convert promutagens into mutagenic metabolites.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
cofactor-supplemented post-mitochondrial S9 fraction
Test concentrations with justification for top dose:
The maximum concentration was 5000 ug/plate (the maximum recommended dose level). In experiment 1, eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate) were assayed in triplicate against each tester strain, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500, 5000 μg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology from plate incorporation to pre-incubation.
Vehicle / solvent:
sterile distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
Details on test system and experimental conditions:
Both the Ames plate incorporation and pre-incubation methods were used.
Rationale for test conditions:
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Consequently, the same maximum dose level was used as the maximum concentration in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method)
Evaluation criteria:
There were several criteria used for determining a positive result. Any, one, or all of the following were used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations
3. Biological relevance against in-house historical control ranges
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989)
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that the program concluded as statistically significant but were within the in-house historical profile were not reported.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
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
Conclusions:
The test item is non-mutagenic in the Ames test both in the presence and in the absence of metabolic activation.
Executive summary:

A GLP Ames test was performed in accordance with current OECD, EU, and EPA testing guidelines. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). Consequently, the same maximum dose level was used as the maximum concentration in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test (pre-incubation method). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic

activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). The positive controls were functional.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 September 2017 to 12 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
For each experiment, whole blood was drawn from the peripheral circulation of a non-smoking male volunteer (28 years old)
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures from PB/βNF induced rats. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.
Test concentrations with justification for top dose:
The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate.
Vehicle / solvent:
Minimal Essential Medium (MEM)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcine (DC)
Details on test system and experimental 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:
8.05-9.05 mL MEM, 10% fetal bovine serum, 0.1 mL Li-heparin, 0.1 mL phytohaemagglutinin, 0.75 mL heparinized whole blood.
- 4-Hour Exposure With Metabolic Activation (S9):
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours
- 4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
- 24-Hour Exposure Without Metabolic Activation (S9)
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours.
The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.
The preliminary toxicity test was performed using the exposure conditions as described for the Main Experiment but using single cultures only, whereas the Main Experiment used replicate cultures.
Rationale for test conditions:
As per the recommendations of the guideline.
Evaluation criteria:
A test item was 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.
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.
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. 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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test substance did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in the 4-hour exposure groups either in the absence or presence of metabolic activation. The test item can therefore be considered to be non-clastogenic and non-aneugenic in these exposure groups.
The 24-hour exposure group did demonstrate small but statistically significant increases over the vehicle control at the three dose levels scored. However, since these increases only marginally exceeded the historical control range for a vehicle, were not part of a dose related response, and were set against a low vehicle control value they were considered to be of no toxicological significance. The response seen at 156 μg/mL in the 24-hour exposure group was within the historical control range for a vehicle. The responses seen at 78 μg/mL and 234 μg/mL were due partly to slightly elevated numbers of micronuclei being seen in only one of the replicate cultures, indicating that the response was not consistent between the replicates and only one of the replicates in each of these dose levels had numbers of micronuclei which exceeded the upper limit of the vehicle historical control.
Remarks on result:
other: Dose limited by precipitate
Conclusions:
The test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
Executive summary:

An in vitro micronucleus test was conducted in human lymphocytes in accordance with the OECD guideline and under GLP conditions.

All vehicle (Minimal Essential Medium) 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 did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei in the 4-hour exposure groups in the presence and absence of S9 using a dose range that included a dose level that was the lowest precipitating dose level. The 24-hour exposure group did demonstrate statistically significant increases in the frequency of binucleate cells with micronuclei but the increases were only marginally greater than the upper limit of the historical control range for a vehicle and were set against a low vehicle control value and were therefore considered to be of no toxicological significance. The upper dose level for the scoring of the 24-hour hour exposure was limited to the lowest precipitating dose level.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 June 2018 to 25 July 2018
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)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
- Type and composition of metabolic activation system: S9 from phenobarbital/beta-naphthone induced male Sprague-Dawley rats
- source of S9 : in-house
- method of preparation of S9 mix: The S9 mix was prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration
- concentration or volume of S9 mix and S9 in the final culture medium: The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Test.
Test concentrations with justification for top dose:
The concentrations used in the main test were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by the onset of test item precipitate, as recommended by the OECD 476 guideline. The concentrations of test item plated for relative survival, cloning efficiency, and expression of mutant colonies were as follows:
2.5, 5, 10, 20, 40, 80 µg/mL for both the experiments with and without S9 mix
Vehicle / solvent:
MEM culture medium
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
Several days before starting each experiment, a fresh stock of cells was removed from the liquid nitrogen freezer and grown up to provide sufficient cells for use in the test. Cells were seeded at 1 x 10e7 cells/225 cm2 flask approximately 24 hours being exposed to the test or control items. This was demonstrated to provide at least 20 x 10e6 cells available for dosing in each flask using a parallel flask. Duplicate cultures were set up, both in the presence and absence of metabolic activation, with eight test item concentrations, and vehicle and positive controls. Treatment was for 4 hours in serum free media (MEM) at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
The concentration range of test item was 1.25 to 160 μg/mL in both the absence and presence of metabolic activation. A repeat of the exposure group in the presence of metabolic activation was performed due to unexpected test item induced toxicity and high vehicle control colony counts.
At the end of the treatment period the flasks were washed twice with PBS, detached from the flasks with trypsin and the cells suspended in MEM with 10% FBS. A sample of each concentration group cell suspension was counted using a Coulter counter. Cultures were plated out at 2 x 10e6 cells/flask in a 225 cm2 flask to allow growth and expression of induced mutants, and in triplicate in 25 cm2 flasks at 200 cells/flask to obtain the cloning efficiency, for an estimate of cytotoxicity at the end of the exposure period. Cells were grown in MEM with 10% FBS and incubated at 37 °C in an incubator with a humidified atmosphere of 5% CO2 in air.
Cytotoxicity flasks were incubated for 7 days then fixed with methanol and stained with Giemsa. Colonies were manually counted to give relative survival (RS). A comparison of the test item to vehicle control relative survivals gave the relative toxicity of each test item dose level.
During the 7 Day expression period the cultures were sub-cultured and maintained on days 2 and 5 to maintain logarithmic growth. At the end of the expression period the cell monolayers were detached using trypsin, cell suspensions counted using a Coulter counter and plated out as follows:
i) In triplicate at 200 cells/25 cm2 flask in 5 mL of MEM with 10% FBS to determine cloning efficiency. Flasks were incubated for 7 days, fixed with methanol and stained with Giemsa. Colonies were manually counted, counts were recorded for each culture and the percentage cloning efficiency for each dose group calculated.
ii) At 2 x 10e5 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. The dishes were incubated for 6 to 7 days at 37 °C in an incubator with humidified atmosphere of 5% CO2 in air, then fixed with methanol and stained with Giemsa. Mutant colonies were manually counted and recorded for each dish.
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:
Test conditions were chosen in accordance with guideline recommendations. The maximum concentration tested was limited by the presence of test item precipitate.
Evaluation criteria:
Provided that all of the acceptability criteria are fulfilled, a test substance is 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 substance concentrations are within the range of the historical negative control data.
Statistics:
Because there was no indication of any increases in mutant frequency at any concentration, a statistical analysis was not necessary.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
True negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: There was no significant change in pH when the test substance was dosed into media
- Data on osmolality: The osmolality did increase by a maximum of 4 mOsm at the concentration levels investigated
- Possibility of evaporation from medium: not reported
- Water solubility: not reported
- Precipitation and time of the determination: At the end of the exposure period, precipitate of the test substance was observed at and above 80 μg/mL in both the absence and presence of metabolic activation .

RANGE-FINDING/SCREENING STUDY: A concentration range of 2.5 to 640 μg/mL was used in the preliminary cytotoxicity test. The maximum concentration tested was limited by the presence of test item precipitate. At the end of the exposure period, precipitate of the test item was observed at and above 80 μg/mL in both the absence and presence of metabolic activation. There was evidence of marked concentration related reductions in cloning efficiency in both the absence and presence of metabolic activation at 160 μg/mL and above. However the toxicity was noted in dose levels with high levels of precipitate. The maximum concentrations selected for the main mutagenicity experiment were therefore limited by the onset of test item precipitate in both the absence and presence of metabolic activation, as recommended by the OECD 476 guideline

STUDY RESULTS
- Concurrent vehicle negative and positive control data : were within the expected ranges.
- Results from cytotoxicity measurements: At the end of the exposure period, the onset of precipitate of the test isubstance was observed at 80 μg/mL in both exposure groups, ie with and without metabolic activation. The Day 0 relative survival and Day 7 cloning efficiencies did not show marked concentration-related reductions in the relative survival values in either the absence or presence of metabolic activation. There was no evidence of any reductions in the Day 7 cloning efficiencies in either the absence or presence of metabolic activation, therefore indicating that residual toxicity had not occurred.
- Genotoxicity results: see attached tables

HISTORICAL CONTROL DATA: provided in study report
Conclusions:
The test substance was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.
Executive summary:

A GLP in vitro gene mutation study in V79 hamster fibroblasts was performed in accordance with the currrent OECD test guideline 475. The test substance did not induce any toxicologically significant or concentration-related increases in the mutant frequency at any of the concentration levels in the main test including the dose level at the onset of test item precipitate in both the absence and presence of metabolic activation. Both of the exposure groups met the requirements recommended by the OECD 476 guideline.

The vehicle (MEM) 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.

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

Additional information

Justification for classification or non-classification

Based on the available data, the test substance does not require classification for mutagenicity according to the CLP Regulation (EC) No 1272/2008