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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): not mutagenic up to cytotoxic concentrations in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100 & TA 102.

- L5178Y/MLA Mammalian Cell Gene Mutation Assay (OECD 490, GLP, K, rel. 1): non mutagenic.

- Micronucleus test in vitro (OECD 487, GLP, K, Rel.1): not clastogenic and not aneugenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 1989-01-18 to 1989-02-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Remarks:
GLP study, comparable to OECD 471 guideline study although not mentioned in the study report
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine gene for Salmonella
Species / strain / cell type:
S. typhimurium, other: TA1535, TA1537, TA1538, TA97, TA98, TA100 and TA102
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital-beta-naphtoflavone-induced rat S9 liver homogenate fraction
Test concentrations with justification for top dose:
0, 1, 5, 10, 25, 50 and 75 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility in DMSO > 50 mg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/2
Positive control substance:
2-nitrofluorene
sodium azide
mitomycin C
other: ICR 191
Remarks:
Without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
See Table 7.6.1/2
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9
Details on test system and experimental conditions:
TEST 1: in agar (plate incorporation)
- Expression time (cells in growth medium): 48 hours at 37°C
- Number of replications: 4 replicate plate for the test material and negative control, two replicate plates for the positive control

TEST 2 : preincubation
- Preincubation period: 30 minutes at 37 °C
- Expression time (cells in growth medium): 48 hours at 37°C
- Number of replications: 4 replicate plate for the test material and negative control, two replicate plates for the positive control

DETERMINATION OF CYTOTOXICITY
- Method: growth of the background lawn and/or frequency of spontaneous revertants (toxicity prescreen on TA100)
Evaluation criteria:
Reproducible, dose-related increase in the number of his+ revertant. This increase should reach at least a doubling of the number of spontaneous revertants for S. typhimurium strains TA1535, TA1537, TA1538 and TA98. For strains TA97, TA100 and TA102, a 1.5-fold increase over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control date;
Statistics:
None
Key result
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA1538, TA97, TA98, TA100 & TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
See "Tables of result" in "Attached background material"
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium:
- Water solubility: Test substance was solubilized in DMSO to improve solubility
- Precipitation: milky suspension at 75 µg/plate
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: was done in TA100, but results were not reported

COMPARISON WITH HISTORICAL CONTROL DATA: Not done

ADDITIONAL INFORMATION ON CYTOTOXICITY: distinct toxic effect were detected in different strains. See "Tables of result" in "Attached background material"

None

Conclusions:
The test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA1538, TA97, TA98, TA100 & TA102.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA 1535, TA 1537, TA1538, TA97, TA 98, TA100 and TA 102 were exposed the test material diluted in DMSO both in the presence and absence of metabolic activation system (phenobarbital / beta-naphtoflavone rat liver homogenate fraction - S9). Plate incorporation assay was used in the first experiment, whereas liquid preincubation assay was performed for the second experiment. The dose range for the main test was determined in a preliminary toxicity assay and was 1 to 75 µg/plate.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused visible reduction in the growth of the bacterial background lawn in the prescreen test and was therefore, tested up to cytotoxicity.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

 

Under the test condition, the test material is not mutagenic with and without metabolic activation in S. thyphimurium strains TA1535, TA1537, TA1538, TA97 TA98, TA100 & TA102.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 February to 26 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD TG 487 without any deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
adopted 29 July 2016
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Programme (inspected on July 05, 2016 / signed on October 26, 2016)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: drawn from the peripheral circulation of a non-smoking volunteer who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection
- Sex, age and number of blood donors if applicable: Preliminary Toxicity Test: Male, aged 33 years; Main Experiment: Female, aged 26 years.
- Normal (negative control) cell cycle time: Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
cytochalasin B (4.5 μg/mL)
Metabolic activation:
with and without
Metabolic activation system:
20% (v/v) S9 fraction; S9 fraction was obtained from the liver homogenates of male rats induced with Phenobarbitone/β-Naphthoflavone
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL. The maximum dose was the maximum practical dose level.
4 h exposure without S9: 0*, 4, 8*, 16*, 24*, 32, 40, 48, 56 µg/mL. The maximum dose was based on toxicity.
4 h exposure with S9 (2%): 0*, 8, 16, 24*, 32*, 40*, 48*, 56, 64 µg/mL. The maximum dose was based on toxicity.
24 h continuous exposure without S9,: 0*, 4*, 8*, 16*, 32*, 40, 48, 56, 64 µg/mL. The maximum dose was based on toxicity.
* Dose levels selected for analysis of micronucleus frequency in binucleate cells.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test item was insoluble in dimethyl sulphoxide at 200 mg/mL but was soluble in acetone at 200 mg/mL but only partially soluble at 400 mg/mL in solubility checks performed in-house.
- Test formulation preparation: Prior to each experiment, the test item was accurately weighed, formulated in acetone and serial dilutions prepared. Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed in 50 μL aliquots. Therefore, the maximum practical dose level was limited to 1000 μg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
other: demecolcine. 0.075 μg/mL in sterile distilled water for 24-hour continuous exposure
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
DURATION
- Exposure duration: 4 h (± S9) and 24 h continuous exposure (-S9) in preliminary toxicity test; 4 h (± S9) and 24 h continuous exposure (-S9) in main experiment
- At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 h in the presence of Cytochalasin B.

CYTOKINESIS INHIBITOR (cytogenetic assays): Prior to the mitosis (after exposure of the test substance) the chemical cytochalasin B (4.5 μg/mL) was added to the cultures.

STAIN (for cytogenetic assays): 5 % Giemsa for 5 minutes

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

NUMBER OF CELLS EVALUATED:
- Cytotoxicity: A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the CBPI value expressed as a percentage of the vehicle controls. The CBPI indicates the number of cell cycles per cell during the period of exposure to Cytochalasin B.
- Scoring of Micronuclei: The micronucleus frequency in 2000 binucleated cells was analyzsed per concentration (1000 binucleated cells per culture, two cultures per concentration) except for CP 5 µg/mL in the presence of S9-mix in the Main Experiment (B culture only) where 2000 cells were assessed for micronuclei. This change to the number of cells assessed was undertaken due to a poor response observed in the first 1000 cells. Cells with 1, 2 or more micronuclei were recorded as such but the primary analysis was on the combined data.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
% Cytostasis = 100-100{(CBPIT – 1)/(CBPIC –1)}
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total number of cells]
T = test substance treatment culture
C = vehicle control culture

OTHER:
The criteria for identifying micronuclei were that they were round or oval in shape, non refractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
- None of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control.
- There was no dose-related increase.
- The results in all evaluated dose groups were within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
- At least one of the test concentrations exhibited a statistically significant increase compared with the concurrent negative control.
- There was an increase which can be considered to be dose-related.
- The results were 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 of a whole chromosome in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. The Study Director may make a judgement based on experience and the biological relevance of the data and any justification for acceptance of the data will be included in the report.
Statistics:
The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cf. Tables of results in attached background documents
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.

PRELIMINARY TOXICITY TEST:
The dose range for the Preliminary Toxicity Test was 0, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 and 1000 μg/mL. The maximum dose was the maximum practical dose level.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at and above 125 μg/mL in the exposure groups in the absence of metabolic activation (S9) and at and above 62.5 μg/mL in the exposure group in the presence of S9.
Hemolysis was observed following exposure to the test item at and above 15.63 μg/mL in the all exposure groups. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes. In addition, a reduced cell pellet was observed at and above 62.5 μg/mL in the 4-hour exposure groups and at and above 125 μg/mL in the 24-hour exposure group.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 31.25 μg/mL in the exposure groups in the absence of S9. The maximum dose with binucleate cells present in the 4-hour exposure group in the presence of S9 was 62.5 μg/mL.
The selection of the maximum dose levels for the Main Experiment were based on toxicity in all three exposure groups.

MAIN EXPERIMENT
The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.
Cf. Table of results in "Attached background documents"

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle control cultures had frequencies of cells with micronuclei within the expected range. 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.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Main experiments:
- The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were binucleate cells suitable for scoring at the maximum dose level of test item, 64 μg/mL in the presence of S9. In the absence of S9, the maximum dose level of the test item with binucleate cells suitable for scoring was 40 and 48 μg/mL in 4-hour and 24-hour exposure groups, respectively.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at 64 μg/mL in the exposure groups in the 24-hour exposure group and the 4-hour exposure in the presence of S9 only.
Hemolysis was observed following exposure to the test item at and above 8 μg/mL in the exposure group in the absence of S9 and at and above 16 μg/mL in the presence of S9. Again, a reduced cell pellet was observed at and above 40 μg/mL in the 4-hour exposure group in the absence of S9 and at and above 56 μg/mL in the 24-hour exposure group. No reduced cell pellet was observed in the presence of S9.
- The CBPI data for the short exposure groups and for the 24-hour exposure group confirm the qualitative observations in that a dose-related inhibition of CBPI was observed in all three exposure groups.
- In the 4-hour exposure group in the absence of S9, 5%, 32% and 78% inhibition of cell proliferation (cytostasis) was observed at 16, 24 and 32 μg/mL. Therefore, the maximum dose level selected for binucleate analysis was 24 μg/mL because the cytostasis value at 32 μg/mL precluded this dose level from analysis due to being in excess of the optimum toxicity range in the OECD 487 guideline (55±5%). Above 32 μg/mL, there were insufficient or no binucleates available for assessment.
- In the 4-hour exposure group in the presence of S9, 14%, 25%, 46% and 71% cytostasis was observed at 32, 40, 48 and 56 μg/mL, respectively. Again, the maximum dose level selected for analysis of binucleate cells was 48 μg/mL because this dose level approached optimum toxicity and the dose level above was in excess of the maximum range for toxicity.
- In the 24-hour exposure group, 19%, 45% and 73% cytostasis was observed at 16, 32 and 40 μg/mL, respectively. The maximum dose level selected for micronuclei analysis was 32 μg/mL as this was approaching optimum toxicity as designated in the guideline. Above 40 μg/mL, there were insufficient or no binucleates available for assessment.

The test item exhibited a steep toxicity curve throughout this study which made achieving optimum toxicity difficult to attain. Therefore, a narrow dose range was used for all three exposure groups in the Main Experiment in an effort to achieve optimum toxicity of 55±5%, as recommended by the OECD 487 Guideline.

In the 4-hour exposure group in the absence of S9, the maximum dose level selected for binucleate analysis was 24 μg/mL, even though the cytostasis value was only 32%. There was an uneven distribution of CPBI between the A and B culture of the solvent control where A was a much lower value than that of the B culture. Had the A culture been similar to the B culture, the resulting cytostasis value would be much closer to optimum. Therefore, it is considered that 24 μg/mL was probably much closer to optimum toxicity than indicated by the reported CPBI values.

In the 4-hour exposure and the 24-hour exposure groups, the cytostasis values for the maximum dose levels selected for binucleate analysis was near optimum but were still slightly below the stated value of 55±5%. However, they were considered to be acceptable as the dose levels were quite narrow and the dose levels above were in excess of optimum toxicity and could not be assessed for micronuclei.

Conclusions:
Under the test conditions, test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to four dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

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 on toxicity. The dose levels selected for the Main Test were as follows:

4-hour without S9: 0, 4, 8, 16, 24, 32, 40, 48, 56 μg/mL

4-hour with S9 (2%): 0, 8, 16, 24, 32, 40, 48, 56, 64 μg/mL

24-hour without S9: 0, 4, 8, 16, 32, 40, 48, 56, 64 μg/mL

 

All vehicle (acetone) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that induced approximately 50% reduction in CBPI.

Under the test conditions, test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 May to 20 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 490 without any deviation.
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
adopted 29 July 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines: - "Kanpoan No. 287 - - Environment Protection Agency" - "Eisei No. 127 - - Ministry of Health and Welfare" - "Heisei 09/10/31 Kikyoku No. 2 - - Ministry of International Trade & Industry"
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Programme (inspected on July 05, 2016 / signed on October 26, 2016)
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK+/- gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
- Suitability of cells: Yes
- Cell cycle length, doubling time or proliferation index: The cells have a generation time of approximately 12 hours and were subcultured accordingly.
- Methods for maintenance in cell culture if applicable: stored in liquid nitrogen at approximately -196°C

MEDIA USED
- Type and identity of media: RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at approximately 37 o C with 5% CO2 in air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
20% S9-mix (i.e. 2% final concentration of S9); S9 was prepared from liver homogenates of male Sprague-Dawley rats orally induced with phenobarbital/β-naphthoflavone (80/100 mg/kg bw/day)
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 3.91, 7.81, 15.63, 31.25, 65.5, 125, 250, 500 and 1000 µg/mL

Mutagenicity test No. 1:
- Without S9-mix (4h exposure): 2, 4, 8, 16, 20, 24, 32, 48 µg/mL
- With S9-mix (4h exposure): 4, 8, 16, 24, 32, 48, 56, 64 µg/mL
- Without S9-mix (24h exposure): 2, 4, 8, 16, 24, 32, 40, 48 µg/mL

Mutagenicity test No. 2:
- With S9-mix (4h exposure): 0, 8, 16, 24, 32, 36, 40, 44, 48, 52 µg/mL
- Without S9-mix (24h exposure): 0, 1, 2, 4, 8, 16, 24, 32, 36, 40, 44 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Test substance preparation: The test item was insoluble in dimethyl sulphoxide at 200 mg/mL but was soluble in acetone at 200 mg/mL, but only partially soluble at 400 mg/mL in solubility checks performed in house.
Prior to each experiment, the test item was accurately weighed, formulated in acetone and dilutions prepared. Due to the sensitivity of the mouse lymphoma cell line to acetone, the formulations were prepared at twice the concentration required to achieve the final concentration in the culture media when dosed in 100 µL aliquots. Therefore, the maximum practical dose level was limited to 1000 µg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Cells were exposed to the test substance in RPMI 1640 with 10% donor horse serum (R10 medium).

DURATION
- Exposure duration: Preliminary test: 4 h (±-S9-mix); 24 h (- S9-mix); Main mutagenicity test: 4 h (±-S9-mix); 24 h (- S9-mix)
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10 to 12 days
- All the incubations were incubated at 37 °C with 5% CO2 in air

SELECTION AGENT (mutation assays): 4 μg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
Preliminary test: single cultures/dose for test item and vehicle control; mutagenicity test: duplicate cultures/dose for test item, positive control and vehicle control

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10E05 cells/mL. The cultures were incubated at 37°C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and diluting to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 10E05 cells/mL in which case all the cells were maintained.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.


DETERMINATION OF CYTOTOXICITY
- Method: Viability, Relative Suspension Growth (RSG)
Evaluation criteria:
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories. Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system. Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
Statistics:
Not required
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cf Tables of results in "Attached background documents"
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality : There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.
- Precipitation: Precipitate of the test item was not observed at any of the dose levels.

RANGE-FINDING/SCREENING STUDIES:
In the all three exposure groups there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The toxicity curve was very steep in both 4-hour exposure groups which would make optimum toxicity difficult to achieve in the main test. A precipitate of the test item was observed at and above 62.5 µg/mL in both 4-hour exposure groups. A precipitate of the test item was observed at and above 500 µg/mL in the 24-hour exposure group. In the subsequent mutagenicity experiments the maximum dose level was limited by test item induced toxicity.

COMPARISON WITH HISTORICAL CONTROL DATA:
The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional

TEST RESULTS:
Cf Tables of results in "Attached background document"

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Main experiment No. 1:
There was evidence of marked toxicity following exposure to the test item in all three exposure groups, as indicated by the %RSG and RTG values. Optimum levels of toxicity were considered to have been achieved in the 4-hour and 24-hour –S9 exposure groups as indicated by the %RSG and RTG values. The toxicity curve was particularly steep between 32 and 48 μg/mL in the 4-hour exposure +S9, 0% and 96% toxicity. However, this response replicated that seen in the preliminary toxicity test and it was expected that optimum toxicity would be hard to achieve. It was decided after consultation with the sponsor, that with the lack of mutagenicity data, due to toxicity, that the 4-hour exposure in the presence of metabolic activation would be repeated to try to achieve optimum toxicity.
There was evidence of reductions in viability (%V) in both 4-hour exposure groups, therefore indicating that residual toxicity had occurred, but only in dose levels with excessive toxicity. There was no evidence of marked reductions in viability in the 24-hour exposure group indicating that residual toxicity had not occurred in this exposure group. It should be noted that the % viability of the vehicle control in the 24-hour exposure group was below the normal limits (65 to 120%) at 56.32 %. This was considered to be due to the toxicity of the acetone over the extended exposure time. The response observed was unusual for a vehicle control. The weak toxic response of acetone was also exhibited in the lower test item dose levels of this group where no test item induced toxicity is apparent. The reduced % viability in the vehicle control group was therefore considered to be unacceptable and a repeat of this exposure group was needed to validate this exposure group.
The dose levels of 48 μg/mL in both the 4 and 24-hour exposure –S9 were not plated out for 5-TFT resistance and viability due to excessive toxicity. The dose levels of 56 and 64 μg/mL in the 4-hour +S9 exposure again were not plated out for 5-TFT resistance and viability due to excessive toxicity. The dose levels of 32 μg/mL in the 4-hour –S9 exposure, 48 μg/mL in the 4-hour +S9 exposure and 40 μg/mL in the 24-hour exposure group were plated out for 5-TFT resistance and %viability as there was sufficient cells for plating. However these dose levels were later excluded from analysis due to excessive toxicity. Acceptable levels of toxicity were seen with both positive control substances. The vehicle controls had mutant frequency values that were considered acceptable for the
L5178Y cell line at the TK +/- locus. However in the 24-hour exposure group the %V vehicle control data was below the normal range indicative of toxicity, which invalidates this exposure group based on acceptability criterion.
The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
The test item did not induce any toxicologically significant increases in the mutant frequency x 10-6 per viable cell in either of the three exposure groups. The GEF value of the test item dose levels were not exceeded in any of the three exposure groups, including dose levels beyond the acceptable level of toxicity in all three exposure groups. No precipitate of the test item was observed at any dose level in the main test. The numbers of small and large colonies and their analysis are presented in Tables 5, 8 and 11. The absence of a dose level with an optimal level of toxicity in the 4 hour exposure group with S9 is considered not to affect the validity of the study because the toxicity dose response was extremely steep, but it was still considered necessary to repeat this exposure using a revised dose range to strengthen the data set.

- Main experiment No. 2: As was seen previously there was evidence of marked toxicity following exposure to the test item in both exposure groups, as indicated by the %RSG and RTG values. In the 4-hour +S9 exposure group, as in the first exposure, optimum levels of toxicity were not quite achieved as indicated by the %RSG and RTG values, even with a 4 μg/mL increments. The toxic nature of the test item proved very difficult to achieve the required 80-90% toxicity. The toxicity curve was steep between 40 and 44 μg/mL in the 4-hour +S9 exposure, 32% and 5% toxicity. It is considered that the 32% survival value recorded at 40 μg/mL was close enough to the desired 20% survival value of the optimum range to satisfy validity of the test. However, this response replicated that seen in the preliminary toxicity test and the first mutagenicity test and it was expected that optimum toxicity would be very hard to achieve. In the 24-hour-S9 exposure group optimum levels of toxicity were
considered to have been achieved as indicated by the %RSG and RTG values (8% RSG 0.24 RTG) (Tables 13 and 16) and the %V for the vehicle control culture (81.33%) was within the desired limits of 65-120%. It was therefore considered that the acceptance criteria had been satisfied.
There was no evidence of marked reductions in viability (%V) in either of the exposure groups, therefore indicating that residual toxicity had not occurred.
The dose levels of 48 and 52 μg/mL in the 4 hour +S9 exposure were not plated out for 5-TFT resistance and viability due to excessive toxicity. The dose levels of 36, 40 and 44 μg/mL in the 24-hour exposure were not plated out for 5-TFT resistance and %viability due to excessive toxicity. The dose level of 44 μg/mL in the 4-hour + S9 exposure was plated out for 5-TFT resistance and %viability as there were sufficient cells for plating. However this dose level was later excluded from analysis due to excessive toxicity. Acceptable levels of toxicity were seen with both positive control substances.
The vehicle controls had mutant frequency and %V values and that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.
The test item did not induce any toxicologically significant increases in the mutant frequency x 10-6 per viable cell in either of the two exposure groups. The GEF value of the test item dose levels were not exceeded in any of the two exposure groups, including a dose level beyond the acceptable level of toxicity (4-hour +S9 44 μg/mL). No precipitate of the test item was observed at any dose level. The numbers of small and large colonies and their analysis are presented in "Attached background document". The absence of a dose level with an optimal level of toxicity in the 4 hour +S9 exposure group is considered not to affect the validity of the study because the toxicity dose response was extremely steep. However the mutagenicity data obtained further strengthens a non-mutagenic response.

Conclusions:
Under the test conditions, test item is not considered as mutagenic at the tk locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.
Executive summary:

In an in vitro mammalian cell gene mutation test performed according to OECD Guideline 490 and in compliance with GLP, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at 8 dose levels in duplicate, together with vehicle (Acetone), and positive controls using 4‑hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-hour exposure group in the absence of metabolic activation.

Two Mutagenicity Tests were performed. The dose range of test item used in the main test No. 1 was selected following the results of a preliminary toxicity test. No precipitate of the test item was observed in the main test. No. 1 The dose levels plated for viability and expression of mutant colonies were as follows:

4-hour without S9: 4, 8, 16, 20, 24, 32 µg/mL

4-hour with S9 (2%): 4, 8, 16, 24, 32, 48 µg/mL

24-hour without S9: 4, 8, 16, 24, 32, 40 µg/mL

The maximum dose level used was limited by test item induced toxicity.  It was decided that with the lack of mutagenicity data, due to excessive toxicity, that the 4-hour exposure in the presence of metabolic activation and the 24 -hour exposure without metabolic activation would be repeated to try to achieve optimum toxicity.

The dose range of test item used in the main test No. 2 was selected following the results of the main test No. 1 alongside the preliminary toxicity test data. No precipitate of the test item was observed in the main test. The dose levels plated for viability and expression of mutant colonies were as follows:

4-hour with S9 (2%): 16, 24, 32, 36, 40, 44, 48, 52 µg/mL

24-hour without S9: 2, 4, 8, 16, 24, 32 µg/mL

The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main tests, in any of the five exposure groups.

 

Under the test conditions, test item is not considered as mutagenic at the TK +/- locus of L5178Y mouse lymphoma cells in the presence and absence of metabolic activation.

 

This study is considered as acceptable and satisfies the requirement for mammalian cells gene mutation endpoint.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

 

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

(Hoffmann, 1989)

Ames Test

(eq. OECD 471)

K, rel. 1

Gene mutation

TA 1535,

TA 1537,

TA 1538,

TA 98,

TA 100

TA 102

-S9

+S9

Up to cytotoxic concentration

-S9 : weak mutagenic

+S9 : weak mutagenic

2

(Envigo, 2018)

L5178YTK+/- MLA test (OECD 490)

K, rel. 1

Gene mutation

L5178Y tk+/-(3.7.2C) mouse lymphoma cells

-S9

+S9

Up to cytotoxic concentration

-S9 : non mutagenic

+S9 : non mutagenic

3

(Envigo, 2017)

MNT vitro

(OECD 487)

K, rel.1

Chromosomal aberration

Human lymphocytes

-S9

+S9

Up to cytotoxic concentration

-S9 and +S9 Not clastogenic

-S9 and +S9 Not aneugenic

Gene mutation Assays (Tests n° 1 and 2):

- A Bacterial Reverse mutation Assay (Ames test) was performed soùilarly to OECD guideline No. 471 with the substance (See Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

- Inability to produce gene mutation was confirmed in mammalian cells using an in vitro gene mutation assay in L5178Y mouse lymphoma cells (L5178Y TK+/- MLA test) (Test n°2). None of the dose levels up to the cytotoxicity limit, either in the presence or absence of metabolic activation, induced significant mutant frequency increases in the initial or repeat experiments whereas both positive control chemicals (with and without metabolic activation) induced significant mutant frequency increases. Therefore, the substance is considered as non-mutagenic and non-aneugenic under the conditions in this assay. This result confirms the results of the Ames test and extends the non-mutagenic effect of the substance to mammalian cells.

 

Chromosomal aberration (Test n°3)

The clastogenic potential of the substance was determined using an in vitro micronucleus test in human lymphocytes (OECD 487), which measures the potential of a substance to increase the incidence of micronuclei in cultured human lymphocytes. The test item was toxic to human lymphocytes but it did not induce any statistically significant increases in the frequency of cells with micronuclei, in any of the exposure groups, using a dose range that included a dose level that induced a sufficient reduction in the cytokinesis block proliferation index (CBPI). Both positive and negative controls validated the sensitivity of the assay. Therefore, the substance is considered as non-clastogenic under the conditions used in this assay.

Justification for classification or non-classification

Harmonized classification:

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

Self classification:

Based on the available data, no self-classification is proposed regarding germ cell mutagenicity according to the CLP and to the GHS.