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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A Klimisch grade 1, GLP compliant bacterial mutation assay performed according to the OECD 471 test method.

A Klimisch grade 1, GLP-compliant mammalian cell chromosome aberration test performed according to the OECD 473 test method.

A Klimisch grade 1, GLP-compliant mammalian cell gene mutation assay performed according to the OECD 490 test method.

The bacterial mutation assay (Ames test), in vitro chromosome aberration test and in vitro mammalian cell gene mutation assay were all negative (non-genotoxic).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
bacterial reverse mutation assay (e.g. Ames test)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 December 2015 to 11 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP in accordance with recognised guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Purity: 100% (UVCB)
- Physical state/Appearance: Yellow semi-solid
- Expiry Date: 23 November 2017
- Storage Conditions: Room temperature in the dark

No correction was made for purity.

The test item was insoluble in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully soluble in dimethyl formamide at the same concentration and acetone at 100 mg/mL in solubility checks performed in-house. Acetone was selected as the vehicle.
The test item was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex mixer and sonication for 10 mins at 40 °C on the day of each experiment. No correction was made for purity. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment 1: Range-finding test: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2: Main test: 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The substance was not sufficiently soluble in water and dimethyl sulphoxide but was soluble in acetone.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(Concurrent - Acetone)
True negative controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
2, 3, 5 µg/plate respectively for WP2uvrA, TA100, TA1535
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(Concurrent - Acetone)
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537
Untreated negative controls:
no
Remarks:
(Concurrent - Acetone)
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
0.2 µg/plate for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(Concurrent - Acetone)
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
1, 2, 10 µg/plate for TA100, TA1535&TA1537, WP2uvrA respectively
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
(Concurrent - Acetone)
True negative controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
5 µg/plate for TA98
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) at multiple dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard/sterilized co-factors).

EXPRIMENT 1
Dose selection
- The maximum concentration was 5000 µg/plate (the maximum recommended dose level).
-Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Without Metabolic Activation
-0.1 mL of the appropriate concentration of test item, solvent vehicle, or appropriate positive control was added to 2 mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate.
- Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described above except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 °C+/- 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
-A number of manual counts were performed due to colonies spreading and bubble interference, disturbing the actual plate count.

EXPERIMENT 2
Dose selection
The dose range used for experiment 2 was determined by the results of experiment 1 and was 5 to 5000 µg/plate.
7 test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item.

Without Metabolic Activation
The procedure was the same as described previously.

With Metabolic Activation
The procedure was the same as described previously.

Incubation and Scoring
All of the plates were incubated at 37 °C +/- 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were performed for count verification purposes due to toxicity.

DURATION
- Preincubation period: N/A
- Exposure duration: Approximately 48 hours
- Expression time (cells in growth medium): N/A
- Selection time (if incubation with a selection agent): N/A
- Fixation time (start of exposure up to fixation or harvest of cells): N/A


SELECTION AGENT (mutation assays): NDA
SPINDLE INHIBITOR (cytogenetic assays): N/A
STAIN (for cytogenetic assays): N/A


NUMBER OF REPLICATIONS: 3 replicates of each strain at each concentration both in the presence and absence of S9

NUMBER OF CELLS EVALUATED:
All strains: 0.9 to 9 *10^9 bacteria/mL

DETERMINATION OF CYTOTOXICITY
- Method: N/A

OTHER EXAMINATIONS:
N/A


OTHER:
Acceptance Criteria
All bacterial strains must have demonstrated the required characteristics as determined by their
respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).

All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:
TA1535: 7 to 40
TA100: 60 to 200
TA1537: 2 to 30
TA98: 8 to 60
WP2uvrA: 10 to 60

Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All
of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation.

There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be 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 will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical analysis of data as determined by UKEMS (Mahon et al, 1989).
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
for TA 1535, TA1537 and TA98 at 5000 µg/plate.
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

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate.The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in the absence of S9-mix, initially from 1500 μg/plate (TA1537) and at 5000 μg/plate (remaining tester strains). In the presence of S9-mix, weakened bacterial background lawns were noted to TA1535, TA98 and TA1537 at 5000 μg/plate. No toxicity was noted to either TA100 or WP2uvrA. Consequently the same maximum dose level (5000 μg/plate) was employed in the second mutation test. A similar toxic response was noted in Experiment 2 with weakened bacterial background lawns noted at 5000 μg/plate to all of the tester strains dosed in the 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. 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.

 

The vehicle (acetone) 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.

Conclusions:
The test item was considered to be non-mutagenic under the conditions of this test.
Executive summary:

INTRODUCTION

The method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline – Bacterial Reverse Mutation Test.

 

METHODS

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to 8 dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 5 to 5000 µg/plate.

 

Seven test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item.

 

RESULTS

The vehicle (acetone) 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 maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in the absence of S9-mix, initially from 1500 µg/plate (TA1537) and at 5000 µg/plate(remaining tester strains). In the presence of S9-mix, weakened bacterial background lawns were noted to TA1535, TA98 and TA1537 at 5000 μg/plate. No toxicity was noted to either TA100 or WP2uvrA. Consequently the same maximum dose level (5000 μg/plate) was employed in the second mutation test. A similar toxic response was noted in Experiment 2 with weakened bacterial background lawns noted at 5000 μg/plate to all of the tester strains dosed in the absence of S9-mix. There was no visible reduction in the growth of the bacterial background lawns of any tester strain dosed in the presence of S9-mix, however small reductions in TA1535 and TA98 revertant colony frequency were noted at the maximum dose level. A light test item film was noted at 5000 μg/plate; this observation did not prevent the scoring of revertant colonies.

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. 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.

 

CONCLUSION

The test substance was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 January 2016 to 30 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP in accordance with recognised guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Physical state: Yellow semi-solid
- Analytical purity: 100%
- Storage condition of test material: Room temperature in the dark
-Expiry date: 23 November 2017
Target gene:
N/A
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum, at approximately 37 °C with 5% CO2 in humidified air.
- The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (HPA).

Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Preliminary Experiment
9.77 to 2500 µg/mL in three exposure groups (4-hour treatment without S9, 4-hour treatment with S9 and 24-hour treatment without S9.
- Test material was soluble in Acetone at 500 mg/mL, however due to toxicity of acetone the dose is reduced to 0.5% and, therefore the maximum achievable dose was reduced to 2500 µg/mL.

Main Experiment
0, 40, 80, 160, 320, 640 and 1280 µg/mL 4-hour treatment without S9
0, 40, 80, 160, 320, 640 and 1280 µg/mL 4-hour treatment with S9
0, 20, 40, 80, 160, 240, 320 and 640 µg/mL 24-hour treatment without S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test item was insoluble at 500 mg/mL in DMSO but was soluble in Acetone at 500 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In absence of S9, 0.2 µg/mL in the 4(20)-hour exposure, 0.1 µg/mL in the 24-hour continuous exposure
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In presence of S9, 2 µg/mL in the 4(20)-hour exposure
Details on test system and experimental conditions:
- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS).
- Properly maintained: NDA
- Periodically checked for Mycoplasma contamination: NDA
- Periodically checked for karyotype stability: NDA
- Periodically "cleansed" against high spontaneous background: NDA

METHOD OF APPLICATION: in medium
With Metabolic Activation (S9 2% final concentration)
Cultures were established approximately 48 hours prior to treatment. Cultures were incubated at 37°C, 5% CO2 in humidified air for 4 hours in the presence of the test material prior to washing. Then, the cells were re-incubated for a further 20 hours without treatment.

Without Metabolic Activation
Cultures were established approximately 48 hours prior to treatment. Cultures were incubated at 37°C, 5% CO2 in humidified air for 24 hours continuous or 4 hours in the presence of the test material prior to washing. For the 4 hours exposure, the cells were then re-incubated for a further 20 hours without treatment.

-The preliminary toxicity test was performed using all three of the exposure conditions as described above for the Main experiment but using single cultures only.
DURATION
- Preincubation period: 48 hours
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 20 or 0 hours
- Fixation time (start of exposure up to fixation or harvest of cells): Mitosis was arrested by addition of democolcine two hours prior to the required harvest time and the cells were harvested, and fixed.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL
STAIN (for cytogenetic assays): 5% Giemsa for 5 minutes

NUMBER OF REPLICATIONS: Treatments performed in duplicate.

NUMBER OF CELLS EVALUATED: Where possible the 300 consecutive well-spread metaphases from each culture (150 per duplicate) were scored for chromosome aberrations.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index was determined by counting a total of 2000 lymphocyte cell nuclei and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes in comparison to controls
- Determination of endoreplication: Yes.
Evaluation criteria:
A test item can be classified as non-genotoxic if:

1. The number of cells with structural aberrations in all evaluated groups is within the range of historical control data.
2. No toxicologically or statistically significant increase in the number of cells with structural chromosome aberrations is observed following statistical analysis.
3. There is no concentration-related increase at any dose level.

A test item can be classified as genotoxic if:

1. The number of cells with structural chromosome aberrations is outside the range of historical control data.
2. At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3. The observed increase in the frequency of cells with structural aberrations is considered to be dose-related.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 9.77, 19.5, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL.
The maximum dose was the maximum practical dose level. A precipitate of the test item was observed at and above 625 µg/mL in the 4(20)-hour exposure groups and at 2500 µg/mL in the continuous exposure group. A greasy/oily precipitate was also observed at 2500 µg/mL in all exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at up to 625 µg/mL in the 4(20)-hour exposure with and without metabolic activation. The maximum dose with metaphases present in the 24-hour continuous exposure was 312.5 µg/mL.
The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level for the 4-hour exposure groups and was toxicity for the the continuous exposure group.

Main Experiment
- Toxicity was similar to that observed in the Preliminary Toxicity test and that there were metaphases suitable for scoring present up to 640 µg/mL in the 4(20)-hour exposure groups with or without S9, and was up to 240 µg/mL in the 24-hour continuous exposure group.
- No precipitate observed in all exposure groups.
- Haemolysis was observed at and above 640 µg/mL in the 4(20)-hour exposure group without S9, at and above 320 µg/mL in the 4(20)-hour exposure group with S9 and at and above 160 µg/mL in the 24-hour continuous exposure group.
- Sligh dose-related inhibition of mitotic index was observed; in the 4(20)-hour exposure group in the presence of S9, 23% mitotic inhibition was achieved at 640 µg/mL.
- In the 4(20)-hour exposure group in the absence of S9, no dose-related inhibition of mitotic index was observed.
- In the 24-hour exposure group, 61% and 73% mitotic inhibition was achieved at 160 and 240 µg/mL respectively.

- The maximum dose level selected for metaphase analysis was 640 µg/mL for the 4(20)-hour exposure in the absence and presence of S9 and 160 µg/mL in the 24-hour exposure.
- Dose level of 240 µg/mL was not scored due to being considered too toxic for metaphase analysis.
- Where this was not achieved dose levels below and above the optimum toxicity limit were selected.

All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that 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 cells with aberrations, either in the absence or presence of metabolic activation. There was no significant increase in the incidence of polyploidy at any dose level in any of the exposure groups. There was no indication of endoreduplication noted.
Remarks on result:
other: negative

See attached background material.

Conclusions:
The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the presence or absence of a liver enzyme metabolizing system. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Introduction

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al., 1990).

 

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9) at a 2% final concentration with cell harvest after 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

 

The dose levels used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (preliminary toxicity test) where the results indicated that the maximum concentration should be limited on precipitate and toxicity. The dose levels selected for the Main Test were as follows.

 

Group                                     Final concentration of test (µg/mL)

4(20)-hour without S9           0, 40, 80, 160, 320, 640, 1280

4(20)-hour with S9 (2%)        0, 40, 80, 160, 320, 640, 1280

24-hour without S9                0, 20, 40, 80, 160, 240, 320, 640

 

Results

All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that 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 cells with aberrations either in the absence or presence of metabolic activation.

 

Conclusion

The test item was considered to be non-clastogenic 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:
08 January 2016 to 02 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed to a recognised guideline and used GLP
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: in vitro mammalian cell gene mutation assay
Specific details on test material used for the study:
- Appearance: Yellow semi-solid
- Analytical purity: 100% (UVCB)
- Expiry date: 23 November 2017
- Storage condition of test material: Room temperature in the dark
Target gene:
The thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:
RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 ug/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 ug/ml) and 10% donor horse serum (giving R10 media) at 37 oC with 5% CO2 in air. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared in-house from the livers of male Sprague-Dawley rats weighing -250g. These had each received, orally, three consecutive daily doses of phenobarbital~-naphthoflavone (80/100 mg per kg per day) prior to S9 preparation on the fourth day.
Test concentrations with justification for top dose:
Preliminary toxicity test 9.77 to 2500 µg/ml for all three of exposure groups.
Main experiment (4-hour exposure) without S9: 0, 23.44, 46.88, 93.75, 187.5, 375, 500, 625, 750 µg/mL
Main experiment (4-hour exposure) with S9: 0, 23.44, 46.88, 93.75, 187.5, 375, 500, 625, 750 µg/mL
Main experiment (24-hour exposure) without S9: 0, 2.5, 5, 10, 20, 40, 60, 80, 100 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone for test item.
- Justification for choice of solvent/vehicle: Not specified.
- Vehicle(s)/solvent(s) used: DMSO for positive controls.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
400 µg/ml in 4-hour exposure group, 150 µg/ml in 24-hour exposure group
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
1.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 ~g/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 ~g/ml) and 10% donor horse serum (giving R10 media).
RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study.
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/mL in 10 ml aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both absence and presence of metabolic activation, and 0.3 x 10^6 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation.
To each universal was added 2 mL of S9 mix if required, 0.1 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).
The cells were exposed to doses of the test material, vehicle and positive control, both with and without metabolic activation. Cultures were maintained at 37 °C in a humidified atmosphere of 5 % CO2 in air.

The treatment regimes were as follows:
DURATION
- Preincubation period: Not applicable.
- Exposure duration: 4 h (Experiment 1 both with and without S9), or 24 h (without S9 Experiment 2).
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10~12 days (plate scoring for colony formation)
SELECTION AGENT (mutation assays): 5-trifluorothymidine (TFT)
STAIN: MTT vital stain for viable cells
NUMBER OF REPLICATIONS: Duplicate
NUMBER OF CELLS EVALUATED: plated 2000 cells/well for mutant frequency; 2 cells/well for viability.
DETERMINATION OF CYTOTOXICITY
- Method: other: Relative Suspension Growth values (RSG)
OTHER: The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.
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:
Dose levels that have survival values less than 10% are excluded from any statistical analysis, as any response they give would be considered to have no biological or toxicological relevance.

The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear-trend events.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Remarks:
strain/cell type: 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:
not examined
Positive controls validity:
valid
Additional information on results:
There was evidence of marked toxicity following exposure to the test item in all three of the exposure groups, as indicated by the %RSG and RTG values (Tables 3, 6, and 9). There was also evidence of modest reductions in viability (%V) in both of the 4-hour exposure groups, indicating that residual toxicity had occurred (Tables 3 and 6, attached). Based on the RTG and %RSG values, it was considered that optimum levels of toxicity were achieved in the 4-hour
exposure group in the presence of metabolic activation, and the 24-hour exposure group in the absence of metabolic activation (Tables 6 and 9, attached). Whilst optimum levels of toxicity were not achieved in the 4-hour exposure group in the absence of metabolic activation due to the very steep toxicity curve of the test item, despite using a very narrow dose range, a dose level that marginally exceeded the upper limit of toxicity based on %RSG was plated as sufficient cells were available at the time of plating. The excessive toxicity observed at and above
625 μg/mL in the 4-hour exposure group in the absence of metabolic activation, at 750 μg/mL in the 4-hour exposure group in the presence of metabolic activation, and at 100 μg/mL in the 24-hour exposure group in the absence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 500 μg/mL in the 4-hour exposure group in the absence of metabolic activation, and at 625 μg/ml in the presence of metabolic activation, exceeded the upper acceptable limit of 90%, therefore, these doses were excluded from analysis. Acceptable levels of toxicity were seen with the positive control substances (Tables 3, 6, and 9, attached).

The test item did not induce any toxicologically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell at any of the dose levels, including the dose levels that achieved optimum levels of toxicity in the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metabolic activation (Tables 6 and 9). The test item also did not induce any toxicologically significant increase in mutant frequency at the dose level in the 4-hour exposure group in the absence of metabolic activation that marginally exceeded the upper limit of acceptable toxicity (Table 3, attached). It should also be noted that the viability plates at this dose level in the 4-hour exposure group in the absence of metabolic activation had acceptable values indicating that the cells had recovered from the initial post-treatment toxicity. The test item was therefore considered to have been adequately tested in all three of the exposure groups. Precipitate of the test item was not observed at any of the dose levels at the end of the exposure period.
Remarks on result:
other:
Remarks:
Non-mutagenic
Conclusions:
The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic under the conditions of this assay.
Executive summary:

INTRODUCTION

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In VitroMammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 28 July 2015, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

 

METHODS

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight 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. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant

colonies were as follows:

 

Mutagenicity Test

Concentration of test item (µg/mL) plated for mutant frequency for the 4-hour without S9 group: 23.44, 46.88, 93.75, 187.5, 375 and 500.

Concentration of test item (µg/mL) plated for mutant frequency for the 4-hour with S9 (2%) group: 46.88, 93.75, 187.5, 375, 500, 625.

Concentration of test item (µg/mL) plated for mutant frequency for the 24-hour without S9 group: 5, 10, 20, 40, 60, 80.

 

RESULTS

The maximum dose level used in the Mutagenicity Test was limited by test item-induced toxicity. Precipitate of the test item was not observed at any of the dose levels at the end of the exposure period in the Mutagenicity Test. 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 test, in any of the three exposure groups.

 

CONCLUSION

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

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

Additional information

IN VITRO BACTERIAL GENE MUTATION ASSAY

INTRODUCTION

The method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 “Bacterial Reverse Mutation Test”, Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008 and the USA, EPA OCSPP harmonized guideline – Bacterial Reverse Mutation Test.

 

METHODS

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to 8 dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 5 to 5000 µg/plate.

 

Seven test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item.

 

RESULTS

The vehicle (acetone) 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 maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 µg/plate. The test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains dosed in the absence of S9-mix, initially from 1500 µg/plate (TA1537) and at 5000 µg/plate(remaining tester strains). In the presence of S9-mix, weakened bacterial background lawns were noted to TA1535, TA98 and TA1537 at 5000 μg/plate. No toxicity was noted to either TA100 or WP2uvrA. Consequently the same maximum dose level (5000 μg/plate) was employed in the second mutation test. A similar toxic response was noted in Experiment 2 with weakened bacterial background lawns noted at 5000 μg/plate to all of the tester strains dosed in the absence of S9-mix. There was no visible reduction in the growth of the bacterial background lawns of any tester strain dosed in the presence of S9-mix, however small reductions in TA1535 and TA98 revertant colony frequency were noted at the maximum dose level. A light test item film was noted at 5000 μg/plate; this observation did not prevent the scoring of revertant colonies.

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. 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.

 

CONCLUSION

The test substance was considered to be non-mutagenic under the conditions of this test.

 

IN VITRO MAMMALIAN CELL CHROMOSOME ABERRATION TEST

INTRODUCTION

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al., 1990).

 

METHODS

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9) at a 2% final concentration with cell harvest after 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

 

The dose levels used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (preliminary toxicity test) where the results indicated that the maximum concentration should be limited on precipitate and toxicity. The dose levels selected for the Main Test were as follows.

 

Group                                     Final concentration of test (µg/mL)

4(20)-hour without S9           0, 40, 80, 160, 320, 640, 1280

4(20)-hour with S9 (2%)        0, 40, 80, 160, 320, 640, 1280

24-hour without S9                0, 20, 40, 80, 160, 240, 320, 640

 

RESULTS

All vehicle (acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that 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 cells with aberrations either in the absence or presence of metabolic activation.

 

CONCLUSION

The test item was considered to be non-clastogenic to human lymphocytes in vitro.

 

IN VITRO MAMMALIAN CELL GENE MUTATION ASSAY

INTRODUCTION

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In VitroMammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 28 July 2015, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

 

METHODS

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight 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. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant

colonies were as follows:

 

Mutagenicity Test

Concentration of test item (µg/mL) plated for mutant frequency for the 4-hour without S9 group: 23.44, 46.88, 93.75, 187.5, 375 and 500.

Concentration of test item (µg/mL) plated for mutant frequency for the 4-hour with S9 (2%) group: 46.88, 93.75, 187.5, 375, 500, 625.

Concentration of test item (µg/mL) plated for mutant frequency for the 24-hour without S9 group: 5, 10, 20, 40, 60, 80.

 

RESULTS

The maximum dose level used in the Mutagenicity Test was limited by test item-induced toxicity. Precipitate of the test item was not observed at any of the dose levels at the end of the exposure period in the Mutagenicity Test. 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 test, in any of the three exposure groups.

 

CONCLUSION

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Justification for classification or non-classification

Harmonized classification:

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

 

Self classification:

Based on the available data, no additional classification for mutagenicity is proposed according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).