[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: 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) and the test item was considered to be non-mutagenic (OECD 471, EU Method B.13/14 and OCSPP harmonized guideline).

 

Chromosome aberration test: The test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolising system. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro (OECD 473, and applicable Japanese guidelines).

 

Mouse lymphoma assay: 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 OECD 490, EU Method B.17, OPPTS 870.5300 and applicable Japanese guidelines).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 June 2016 to 24 June 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

S9 fraction pre-prepared in bulk with no impact on validity or integrity of study (see below)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

S9 fraction pre-prepared in bulk with no impact on validity or integrity of study (see below)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

yes

Remarks:

S9 fraction pre-prepared in bulk with no impact on validity or integrity of study (see below)

Qualifier:

according to guideline

Guideline:

other: OCSPP harmonized guideline - Bacterial reverse mutation assay

Deviations:

yes

Remarks:

S9 fraction pre-prepared in bulk with no impact on validity or integrity of study (see below)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
- Experiment 2: 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

Acetone

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Untreated negative controls:

yes

Remarks:

untreated controls

Positive controls:

yes

Remarks:

without metabolic activation (2 µg/plate for WP2uvrA; 3 µg/plate for TA100; 5 µg/plate for TA1535)

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

ENNG; direct acting compound; batch 67F-3700 treated as 100 % despite no data sheet being available; expiry date 18 September 2017; solvent DMSO

Positive controls:

yes

Remarks:

without metabolic activation (80 µg/plate for TA1537)

Positive control substance:

9-aminoacridine

Remarks:

9AA; direct acting compound; batch S32398-438; purity 99.9 %; expiry date 01 October 2017; solvent DMSO

Positive controls:

yes

Remarks:

without metabolic activation (0.2 µg/plate for TA98)

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

4NQO; direct acting compound; batch 030M1206; purity 100 %; expiry date 08 October 2017; solvent DMSO

Positive controls:

yes

Remarks:

with metabolic activation (1 µg/plate for TA100; 2 µg/plate for TA1535 and TA1537; 10 µg/plate for WP2uvrA)

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA; indirect acting compound; batch STBB1901M9; purity 97.5 %; expiry date 08 October 2017; solvent DMSO

Positive controls:

yes

Remarks:

with metabolic activation (5 µg/plate for TA98)

Positive control substance:

benzo(a)pyrene

Remarks:

BP; indirect acting compound; batch 090M1400V; purity 96 %; solvent DMSO

Details on test system and experimental conditions:

STUDY CONTROLS
- Acetone was used as the solvent (vehicle) control.
- Negative (untreated) controls were performed to assess the spontaneous revertant colony rate.
- Solvent and negative controls were performed in triplicate.
- The positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation.
- Positive controls were performed in triplicate.
- Sterility controls were performed (i) top agar and histidine/biotin or tryptophan in the absence of S9 mix in triplicate (ii) top agar and histidine/biotin or tryptophan in the presence of S9 mix in triplicate (iii) singular test of maximum dosing solution of the test item in the absence of S9 mix only.

MICROSOMAL ENZYME FRACTION
- The S9 Microsomal fractions were pre-prepared using standardized in-house procedures.
- Lot No. PB/βNF S9 10 April 2016 was used in the study.
- A copy of the S9 Certificate of Efficacy is presented in Appendix 2 (attached).

S9-MIX AND AGAR
- The S9-mix was prepared before use using sterilised co-factors and maintained on ice for the duration of
the test.
- Composition of the S9-mix was S9 (5.0 mL), 1.65 M KCl/0.4 M MgCl2 (1.0 mL), 0.1 M glucose-6-
phosphate (2.5 mL), 0.1 M NADP (2.0 mL), 0.2 M sodium phosphate buffer pH 7.4 (25.0 mL) and sterile
distilled water (14.5 mL).
- An aliquot (0.5 mL) of S9-mix and molten, trace histidine or tryptophan supplemented, top agar (2 mL)
were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix.
This procedure was repeated in triplicate on the day of the experiment.

MEDIA
- Top agar was prepared using 0.6% Bacto agar (lot number 5054857 12/19) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar.
- Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 41720 06/16 and 41792 07/16).

BACTERIA
- Details of the five strains of bacteria used and their mutations are attached.
- All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally, due to the "deep rough" (rfa-) mutation, they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin.
- In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000).
- In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
- Bacteria used in the test were obtained from University of California, Berkeley, on culture discs (04 August 1995) and British Industrial Biological Research Association, on a nutrient agar plate (17 August 1987).
- All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer (model SXR 34).
- In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot numbers 1712138 07/20 and 1758279 10/20) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.
TEST ITEM PREPARATION AND ANALYSIS
- During solubility checks performed in-house the test item was noted to be immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but fully soluble in acetone at the same concentration in solubility checks performed in-house. Acetone was therefore selected as the vehicle.
- The test item was accurately weighed and approximate half-log dilutions prepared in acetone by mixing on a vortex mixer on the day of each experiment. No correction was made for purity.
- Prior to use, the solvent was dried to remove water using molecular sieves (2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns).
- 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.

EXPERIMENT 1 – DOSE SELECTION
- 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.
- The direct plate incorporation method was used.

EXPERIMENT 1 – WITHOUT METABOLIC ACTIVATION
- An aliquot (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.
- The components were mixed and overlaid onto a Vogel-Bonner agar plate.
- Negative (untreated) controls were also performed on the same day as the mutation test.
- Each concentration of the item, appropriate positive, vehicle and negative controls was assayed in triplicate for each bacterial strain.

EXPERIMENT 1 – WITH METABOLIC ACTIVATION
- The procedure was the same as described previously except that, following the addition of test item formulation and bacterial culture, S9-mix (0.5 mL) was added to the molten, trace amino acid supplemented media instead of phosphate buffer.

EXPERIMENT 1 – INCUBATION AND SCORING
- All of the plates were incubated at 37 ± 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 required, predominantly due to interference in the base agar (such as minor precipitation of salts/dust particles slightly distorting the counts).

EXPERIMENT 2 – DOSE SELECTION
- The procedure was repeated using the plate incorporation method in the presence and absence of metabolic activation.
- The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15 to 5000 μg/plate.
- Six test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item.

EXPERIMENT 2 – WITHOUT METABOLIC ACTIVATION
- The procedure was the same as described for Experiment 1.

EXPERIMENT 2 – WITH METABOLIC ACTIVATION
- The procedure was the same as described for Experiment 1.

EXPERIMENT 2 – INCUBATION AND SCORING
- All of the plates were incubated at 37 ± 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).

ACCEPTABILITY CRITERIA
- The reverse mutation assay may be considered valid if the criteria given below are met.
- All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks (Ames et al, 1975; Maron and Ames, 1983; 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 7 to 40 (TA1535); 60 to 200 (TA100); 2 to 30 (TA1537); 8 to 60 (TA98); 10 to 60 (WP2uvrA).
- Combined historical negative and solvent control ranges for 2014 and 2015 are presented in Appendix 1 (attached).
- All tester strain cultures should be in the range of 0.9 to 9 x 10E09 bacteria per mL.
- 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 (with and without metabolic activation). The historical ranges of the positive control reference items for 2014 and 2015 are presented in Appendix 1 (attached).
- There should be a minimum of four non-toxic test item dose levels.
- There should be no evidence of excessive contamination.

MAJOR COMPUTERISED SYSTEMS
- Ames Study Manager and Sorcerer Imaging System.
- Delta Building Monitoring System.

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:
(a) A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
(b) A reproducible increase at one or more concentrations.
(c) Biological relevance against in-house historical control ranges.
(d) Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
(e) 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 significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

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

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTATION TEST
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate. All were found to be satisfactory.
- The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data were not given in the report.
- Results for the negative controls (spontaneous mutation rates) are presented in Table 1 (attached) and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
- The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and vehicle controls, both with and without metabolic activation, are presented in Tables 2 and 3 (attached) for Experiment 1 and Table 4 and 5 for Experiment 2 (attached).
- A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 1 (attached).
- The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate.
- In the first mutation test, there was no visible reduction in the growth of the bacterial background lawn a t any dose level, either in the presence or absence of metabolic activation (S9-mix). The same maximum dose level was therefore used in the second mutation test.
- Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test.
- No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
- In Experiment 1, 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).
- Similarly, no toxicologically 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.
- Small, statistically significant increases in TA1537 revertant colony frequency were observed in the second mutation test at 5000 μg/plate (absence of S9-mix only). This increase was considered to be of no biological relevance because there was no evidence of reproducibility or a dose-response relationship. Furthermore, the individual revertant counts at the statistically significant dose level were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.6 times the
concurrent vehicle control.
- 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 the test.

Executive summary:

GUIDELINE

The test 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 atup to eight dose levels, in triplicate, both with and without the addition of a rat liverhomogenate metabolizing system (10% liver S9 in standard co-factors). The dose range forExperiment 1 was predetermined and was 1.5 to 5000μg/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 15 to 5000 μg/plate. Six test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the potential 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. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

 

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no toxicologically 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. Small, statistically significant increases in TA1537 revertant colony frequency were observed in the second mutation test at 5000 μg/plate (absence of S9-mix only). This increase was considered to be of no biological relevance because there was no evidence of reproducibility or a dose-response relationship. Furthermore, the individual revertant counts at the statistically significant dose level were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.6 times the concurrent vehicle control.

 

CONCLUSION

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

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 June 2016 to 21 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: chromosome aberration

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes:

Remarks:

human

Details on mammalian cell type (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 % foetal bovine serum (FBS) at approximately 37 °C with 5 % CO2 in humidified air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by addition of phytohaemagglutinin (PHA).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (see Appendix 2, attached)

Test concentrations with justification for top dose:

PRELIMINARY TEST
0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 µg/mL

MAIN TEST
- 4(20) hour without S9: 0, 2.5, 5, 10, 15, 20, 40, 80 µg/mL
- 4 (20) hour with S9 (2 %): 0, 5, 10, 15, 20, 40, 80, 160 µg/mL
- 24 hour without S9: 0, 2.5, 5, 10, 15, 20, 40, 80 µg/mL

Vehicle / solvent:

Acetone

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

Remarks:

Fisher Scientific batch 1664439 (expiry date 01/2021)

Positive controls:

yes

Remarks:

4 hour exposure in the absence of S9

Positive control substance:

mitomycin C

Remarks:

Sigma Aldrich batch SLBM6258V, purity 100 % (expiry date 01 May 2019) 0.2 µg/L

Positive controls:

yes

Remarks:

4 hour exposure in the presence of S9

Positive control substance:

cyclophosphamide

Remarks:

Acros Organics batch A0355340, purity 97 % (expiry date 01 January 2020) 5 µg/L

Details on test system and experimental conditions:

CELLS
- For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (aged 18-35) who had previously been screened for suitability.
- The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly suffered from a recent viral infection.
- Donor used for the preliminary test was male, aged 25 years.
- Donor used for the main experiment was female, aged 28 years.
- 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.

MICROSOMAL ENZYME FRACTION AND S9 MIX
- The S9 microsomal fractions were pre-prepared using standardised in-house procedures.
- Lot PB/ßNF S9 10/04/16 was used in the study.
- A copy of the S9 certificate of efficacy is given in Appendix 2 (attached).
- The S9-mix was prepared prior to dosing of the test cultures and contained the S9 fraction (20 % v/v), MgCl2 (8 mM), KCl (33 mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM) and NADP (5 mM).
- The final concentrations of S9 was 2 % when dosed at 10 % volume of S9-mix into culture media.

TEST ITEM PREPARATION AND ANALYSIS
- The test item was considered to be a UVCB and the maximum recommended dose was therefore initially set at 5000 µg/mL.
- The purity of the test item was considered to be 100 % and was not accounted for in the test item formulations.
- Solubility checks performed in-house showed the test material was immiscible in dimethyl sulphoxide at 500 mg/mL but was fully miscible in acetone at the same concentration.
- 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. The maximum practical concentration was consequently 2500 µg/mL.
- Prior to each experiment, the test item was accurately weighed, formulated in acetone and appropriate serial dilutions were prepared.
- There was no significant change in pH when the test item was dosed into media and the osmolarity did not increase by more than 50 mOsm. The pH and osmolarity readings are presented in the table below.
- The test item was formulated within two hours of it being applied to the test system and formulations were assumed to be stable. No analysis was conducted to determine homogeneity, concentration or stability of the test item formulations because it is not a requirement of the test guideline. This is an exception with regard to GLP and was reflected in the GLP compliance statement.

CULTURE CONDITIONS
- Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing MEM, 10 % FBS (9.05 mL), Li-heparin (0.1 mL), phytohaemagglutin (0.1 mL) and heparinised whole blood (0.75 mL).

4-HOUR EXPOSURE WITH METABOLIC ACTIVATION (S9)
- After approximately 48 hours incubation at approximately 37 °C and 5 % CO2 in humidified air, the cultures were transferred to tubes and centrifuged.
- Approximately 9 mL of the culture medium was removed and replaced with the required volume of MEM (including serum) and 0.05 mL of the appropriate solution of vehicle control or test item was added to each culture.
- For the positive control, the appropriate solution (0.1 mL) was added to the cultures.
- S9-mix 20 % (1 mL) was added to the cultures of the preliminary toxicity test and main experiment to give a final concentration of 2 % S9 in standard co-factors.
- After 4 hours at approximately 37 °C and 5 % CO2 in humidified air, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium.
- After a further centrifugation, the wash medium was removed by suction and replaced with the original culture medium.
- The cells were then re-incubated for a further 20 hours at approximately 37 °C and 5 % CO2 in humidified air.

4-HOUR EXPOSURE WITHOUT METABOLIC ACTIVATION
- After approximately 48 hours incubation at approximately 37 °C with 5 % CO2 in humidified air, the cultures were decanted into tubes and centrifuged.
- Approximately 9 mL of the culture medium was removed and reserved.
- The cells were then re-suspended in the required volume of fresh MEM (including serum) and dosed with 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution.
- The total volume for each culture was a nominal 10 mL.
- After 4 hours at approximately 37 °C and 5 % CO2 in humidified air, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium.
- After a further centrifugation, the wash medium was removed by suction and replaced with the reserved original culture medium.
- The cells were then returned to the incubator for a further 20 hours.

24-HOUR EXPOSURE WITHOUT METABOLIC ACTIVATION
- As the exposure was continuous the cultures were established at a nominal volume of 9.9 mL.
- After approximately 48 hours incubation, the cultures were removed from the incubator and dosed with 0.05 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution.
- The nominal final volume of each culture was 10 mL.
- The cultures were then incubated at approximately 37 °C and 5 % CO2 in humidified air for 24 hours.
- The preliminary toxicity test was performed using all three of the exposure conditions as described for the main experiment but using single cultures only.

PRELIMINARY TOXICITY TEST
- Three exposure groups were used.
(i) 4-hour exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media; 4(20)-hour exposure.
(ii) 4-hour exposure to the test item with S9-mix (2 %), followed by a 20-hour recovery period in treatment-free media; 4(20)-hour exposure.
(iii) 24-hour continuous exposure to the test item without S9-mix.
- Parallel flasks containing culture medium without whole blood were established for the three exposure conditions so that test item precipitate observations could be made.
- Precipitate observations were recorded at the beginning and end of the exposure periods.
- Using a qualitative microscopic examination of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation.
- Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the main test.

MAIN EXPERIMENT
- Three exposure groups were used for the main experiment.
(i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest.
(ii) 4-hour exposure to the test item with S9-mix (2 %), followed by 20-hour culture in treatment-free media prior to cell harvest.
(iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest.
- Parallel flasks containing culture medium without whole blood were established for the three exposure conditions so that the test item precipitate observations could be made.
- Precipitate observations were recorded at the beginning and end of the exposure periods.

CELL HARVEST
- Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time.
- After incubation with demecolcine, the cells were centrifuged, culture medium was drawn off and discarded, and the cells were re-suspended in 0.075 M hypotonic KCl.
- After approximately 14 minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded.
- The cells were re-suspended and then fixed by dropping the KCl suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 °C to ensure complete fixation prior to slide preparation.

PREPARATION OF METAPHASE SPREADS
- The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative.
- Several drops of the suspension were dropped onto clean, wet, microscope slides and left to air dry.
- each slide was permanently labelled with appropriate identification data.

STAINING
- When the slides were dry they were stained in 5 % Giemsa for 5 minutes.
- The slides were then rinsed and dried before a cover slip was applied using mounting medium.

QUALITATIVE SLIDE ASSESSMENT
- Slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of test item precipitation.
- These observations were used to select the dose levels for mitotic index evaluation.

CODING
- The slides were coded using a computerised random number generator.
MITOTIC INDEX
- A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase was recorded.
- The number if cells in metaphase was expressed as the mitotic index and as a percentage of the vehicle control value.

SCORING of CHROMOSOME DAMAGE
- Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate).
- Where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated.
- If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985) (Appendix 1).
- Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
- In addition, cells with 60 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) including the incidence of cells with endoreduplicated chromosomes was also reported.
- Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors and the current historical range was shown in Appendix 1 (attached).

CRITERIA TO DETERMINE A VALID ASSAY
- The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures should be within the laboratory historical control data range.
- All the positive control chemicals should induce a positive response (p ≤ 0.01) and demonstrate the validity of the experiment and the integrity of the S9-mix.
- The study should be performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
- The required number of cells and concentrations should be analysed.

MAJOR COMPUTERISED SYSTEMS
- Data analysis was performed using a program developed in-house.
- Delta Building Monitoring System.

Evaluation criteria:

CRITERIA FOR DETERMINING THE STUDY CONCLUSION
- Providing that all the acceptability criteria were fulfilled, a test item could be considered to be clearly negative if, in any of the experimental conditions examined:
(1) The number of cells with structural aberrations in all evaluated dose groups was within the range of the laboratory historical control data.
(2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations was observed following statistical analysis.
(3) There was no concentration-related increase at any dose level.
- A test item could be considered genotoxic if:
(1) The number of cells with structural chromosome aberrations was outside the range of the laboratory historical control data.
(2) At least one concentration exhibited 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 was considered to be dose-related.
- When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
- Although inclusion of the structural chromosome aberrations was the purpose of the study, it was important to include numerical aberrations in the form of polyploidy and endoreduplicated cells.

Statistics:

STATISTICAL ANALYSIS
- 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 (Richardson et al, 1989).
- A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps was less than 0.05 compared to its concurrent control and there was a dose-related increase in the frequency of cells with aberrations which was reproducible.
- Incidences where marked statistically significant increases were observed only with gap-type aberrations was assessed on a case by case basis.

Species / strain:

other: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating 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 to 2500 µg/mL.
- The test item was considered to be a UVCB and therefore the maximum recommended dose was initially set at 5000 µg/mL.
- The test item was immiscible in dimethyl sulphoxide at 500 mg/mL but was fully miscible in acetone at the same concentration during solubility checks performed in-house.
- 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.
- The maximum practical concentration was therefore 2500 µg/mL.
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of exposure at and above 39.06 µg/mL (without metabolic activation) and at and above 79.13 µg/mL (with S9).
- Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to the maximum dose level of 2500 µg/mL in all three exposure groups.
- Mitotic index data are presented in Table 1 (attached).
- The test item induced no evidence of toxicity in any of the exposure groups.
- Selection of the maximum dose level for the main experiment was therefore based on the lowest precipitating dose level for all three exposure groups.

MAIN EXPERIMENT
- Dose levels of the controls and test item are shown in the table below.
- the qualitative assessment of the slides determined that the precipitate was similar to that observed in the preliminary toxicity test and that there were metaphases suitable for scoring present up to the maximum dose level tested in all three exposure groups.
- Precipitate observations were made at the end of exposure in blood-free cultures and were noted at and above 40 µg/mL in the 4-hour exposure group without S9, at and above 80 µg/mL in the 4-hour exposure group in the presence of S9 and at and above 20 µg/mL in the 24-hour continuous exposure group.
- The mitotic index data for the main experiment are given in Table 2 (attached) and Table 3 (attached). They confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in any of the exposure groups.
- The maximum dose level selected for the metaphase analysis was therefore the lowest precipitating dose level, which was 40 µg/mL in the 4-hour exposure group without S9, 80 µg/mL in the 4-hour exposure group in the presence of S9 and 20 µg/mL in the 24-hour continuous exposure group.
- Chromosome aberration data are given in Tables 4, 5 and 6 (attached).
- The assay was considered valid because the following criteria were met:
(i) Frequency of sells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within historical control data range
(ii) All the positive control chemicals induced a demonstrable positive response (p ≤ 0.01) and confirmed the validity and sensitivity of the assay plus the integrity of the S9-mix.
(iii) The study was performed using all three exposure conditions and a top concentration which meets the requirements of the current testing guideline.
(iv) The required number of cells and concentrations were analysed.
(v) 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.
(vi) The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups (see Table 7, attached).

Dose levels of the controls and test item

Group	Final concentration of test item (µg/mL)
4(20)-hour without S9	0*, 2.5, 5, 10*, 15*, 20*, 40*, 80, MMC 0.2*
4(20-hour with S9 (2 %)	0*, 5, 10, 15*, 20*, 40*, 80*, 160, CP 2*
24-hour without S9	0*, 2.5, 5*, 10*, 15*, 20*, 40, 80, MMC 0.1*

* Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

Conclusions:

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

Executive summary:

GUIDELINE

Structural chromosomal aberrations were investigated in cultured mammalian cells in accordance with OECD Guidelines for Testing of Chemicals No 473 "In Vitro Mammalian Chromosome Aberration Test" adopted 26 September 2014 and the Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI) and Ministry of the Environment (MOE) Guidelines of 31 March 2011.

METHODS

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. The study investigated three exposure conditions: 4 hours exposure in the presence of an induced rat liver homogenate metabolising system (S9) at a 2 % final concentration with cell harvest a 20 hour expression period; 4 hours exposure in the absence of metabolic activation (S9) with a 20 hour expression period; 24 hour exposure in the absence of metabolic activation. The dose levels used in the main experiment were selected using data from the preliminary toxicity test where results indicated that the maximum concentration should be limited on precipitate. The dose levels used in the main test were 0, 2.5, 5, 10, 15, 20, 40, 80 µg/L for 4(20) hour without S9; 0, 5, 10, 15, 20, 40, 80, 160 µg/L for 4(20) hour with S9 (2 %); 0, 2.5, 5, 10, 15, 20, 40, 80 µg/L for 24 hour without S9.

RESULTS

All vehicle (acetone) controls had frequencies of cells with aberration within the range expected for normal human lymphocytes. All the positive controls induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated. The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations using a dose range that included a concentration that was the lowest precipitating dose level.

CONCLUSION

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

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August 2016 to 06 September 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

yes

Remarks:

S9 pre-prepared in bulk with no impact on integrity or outcome of the study (see below)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

S9 pre-prepared in bulk with no impact on integrity or outcome of the study (see below)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

yes

Remarks:

S9 pre-prepared in bulk with no impact on integrity or outcome of the study (see below)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mouse lymphoma assay

Target gene:

Thymidine kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital / ß-Naphtha flavone induced S9 mix (see Appendix 2, attached)

Test concentrations with justification for top dose:

- Preliminary cytotoxicity test: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL
- Main experiment: 1.25, 2.5, 5, 10, 20, 40 µg/mL (4-hour without S9 and 4-hour with 2 % S9)
- Main experiment: 2.5, 5, 10, 20, 40, 80 µg/mL (24-hour without S9).

Vehicle / solvent:

Acetone

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Remarks:

EMS in DMSO for use without metabolic activation (400 μg/mL for 4-hour exposure and 150 μg/mL for 24-hour exposure)

Positive control substance:

ethylmethanesulphonate

Remarks:

Sigma (batch BCBQ0451V; purity 100 %; expiry date 12.02.17)

Positive controls:

yes

Remarks:

CP in DMSO for use with metabolic activation (1.5 μg/mL)

Positive control substance:

cyclophosphamide

Remarks:

Acros Organics (batch A0355340; purity 97 %; expiry date 24.11.17)

Details on test system and experimental conditions:

STUDY PURPOSE
- 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 use of cultured mammalian cells for mutation studies may give a measure of the intrinsic response of the mammalian genome and its maintenance process to mutagens. Such techniques have been used for many years with widely different cell types and loci. The thymidine kinase heterozygote system, TK +/- to TK -/-, was described by Clive et al., (1972) and is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). This
system has been extensively validated (Clive et al., 1979; Amacher et al., 1980; Jotz and Mitchell, 1981).
- The technique used was a fluctuation assay using microtitre plates and trifluorothymidine as the selective agent and is based on that described by Cole and Arlett (1984). Two distinct types of mutant colonies can be recognised, i.e. large and small. Large colonies grow at a normal rate and represent events within the gene (base-pair substitutions or deletions) whilst small colonies represent large genetic changes involving chromosome 11b (indicative of clastogenic activity).

CELL LINE
- 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.

CELL CULTURE
- The stocks of cells are stored in liquid nitrogen at approximately -196 °C.
- Cells were routinely cultured in 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 37 °C with 5% CO2 in air.
- The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), were used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

MICROSOMAL ENZYME FRACTION
- Lot No. PB/βNF S9 10/04/16 was used in this study, and was pre-prepared in-house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test and a certificate of S9 efficacy is presented in Appendix 2 (attached).
- The S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.
- A concentration of 20 % S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and Mutagenicity Test.

CELL CLEANSING
- The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but
significant rate. Before the stocks of cells were frozen they were cleansed of homozygous
(TK -/-) mutants by culturing in THMG medium for 24 hours.
- The THMG medium contained Thymidine (9 μg/mL), Hypoxanthine (15 μg/mL), Methotrexate (0.3 μg/mL) and Glycine (22.5 μg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

TEST ITEM PREPARATION
- Following solubility checks performed in-house for the Chromosome Aberration Test performed on the same material, the test item was accurately weighed and formulated in acetone prior to serial dilutions being prepared.
- The test item was considered to be a complex mixture (UVCB) therefore the maximum proposed dose level in the solubility test was set at 5000 μg/mL, the maximum recommended dose level, and no correction for the purity of the test item was applied. However, acetone is toxic to L5178Y cells at dose volumes greater than 0.5% of the total culture volume. Therefore, the test item was formulated at 500 mg/mL and dosed at 0.5% to give a maximum achievable dose level of 2500 μg/mL. There was no marked change in pH when the test item was dosed into media
and the osmolality did not increase by more than 50 mOsm (Scott et al. 1991).
- The pH and osmolality readings from the chromosome aberration test are given in the table below.
- No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and was reflected in the GLP compliance statement.

PRELIMINARY TOXICITY TEST
- A preliminary toxicity test was performed on cell cultures at 5 x 10E05 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 9.77 to 2500 μg/mL for all three of the exposure groups. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL
in which case all the cells were maintained.
- The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10E05 cells/mL, unless the mean cell count was less than 3 x 10E05 cells/mL in which case all the cells were maintained. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post
exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.
- Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 μL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

MUTAGENICITY TEST
- 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 10E06 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10E06 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.
- The exposures were performed in duplicate (A + B), both with and without metabolic activation (2 % S9 final concentration) at eight dose levels of the test item (0.63 to 80 μg/mL for all three of the exposure groups), vehicle and positive controls. 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 exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

MEASUREMENT OF SURVIVAL, VIABILITY AND MUTANT FREQUENCY
- At the end of the exposure periods, 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 dilution to 2 x 10E05 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 10E04 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.
- The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure 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.

PLATE SCORING
- Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37°C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al., 1990).
- Colonies were scored manually by eye using qualitative judgment. Large colonies are defined as those that cover approximately 0.25 to 0.75 of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small
colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualization of the mutant colonies, particularly the small colonies.

CALCULATION OF PERCENTAGE RELATIVE SUSPENSION GROWTH (%RSG)
- The cell counts obtained immediately post exposure and over the 2-day expression period were used to calculate the Percentage Relative Suspension Growth:
(i) 4-Hour Suspension Growth (SG) = (24-hour cell count/2) x (48-hour cell count/2)
(ii) 24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) x (24-hour cell count/2) x (48 hour cell count/2)
(iii) Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
(iv) %RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100
CALCULATION OF DAY 2 VIABILITY (%V)
- Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method:
(i) P(0) = number of negative wells / total wells plated
(ii) %V = -ln P(0) x 100 / number of cells/well

CALCULATION OF RELATIVE TOTAL GROWTH (RTG)
- For each culture, the relative cloning efficiency, RCE, was calculated:
(i) RCE = %V / mean solvent control %V
(ii) RTG = (RCE / RSG) x 100

DATA EVALUATION
- The current Historical Vehicle and Positive Control Mutation Frequencies are presented in Appendix 1 (attached). The Historical Vehicle and Positive Control data is generated by the Mutant 240C program on a rolling system of the last twenty sets of archived data. The program combines the 4-hour and 24-hour data in the absence of metabolic activation as the acceptability criteria is the same for all three of the exposure groups.
- Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
- 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 10E-06, 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.

ACCEPTABILITY OF ASSAY
- A mutation assay is considered acceptable if it meets the following criteria (the current recommendations of the IWGT will be considered):
(1) The majority of the plates, for both viability (%V) and TFT resistance, are analysable for each experiment.
(2) The absolute viability (%V) at the time of mutant selection of the solvent controls is 65 to 120 %.
(3) The total suspension growth of the solvent control following 4 hour exposure, calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold increase in cell number, should be in the range of 8 to 32. Following 24 hour exposure the total suspension growth should be in the range of 32 to 180.
(4) The in-house vehicle control mutant frequency is in the range of 50 – 170 x 10E-06 cells. Vehicle control results should ideally be within this range, although minor errors in cell counting and dilution, or exposure to the metabolic activation system, may cause this to be slightly elevated. Experiments where the vehicle control values are markedly greater than 200 x 10E-06 mutant frequency per survivor are not acceptable and will be
repeated.
(5) Every test should also be evaluated as to whether the positive controls (EMS and CP) meets at least one of the following two acceptance criteria developed by the IWGT workgroup:
(a) The positive control should demonstrate an absolute increase in total MF, that is, an increase above the spontaneous background MF [an induced MF (IMF)] of at least 300 x 10E-06. At least 40% of the IMF should be reflected in the small colony MF.
(b) The positive control has an increase in the small colony MF of at least 150 x 10E-06 above that seen in the concurrent untreated/solvent control (a small colony IMF of 150 x 10E-06).
(6) The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures i.e. the Relative Total Growth (RTG) and percentage Relative Suspension Growth (%RSG) should be greater than approximately 10 % of the concurrent selective control group.
(7) For non-toxic test items the upper test item concentrations will be 10mM, 2 mg/mL or 2μL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCBs) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL. Precipitating dose levels will not be tested
beyond the onset of precipitation regardless of the presence of toxicity beyond this point. In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival.

Evaluation criteria:

See above

Statistics:

CALCULATION OF MUTATION FREQUENCY (MF)
- The mutation frequency (MF) per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.
- 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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY CYTOTOXICITY TEST
- The dose range of the test item used in the preliminary toxicity test was 9.77 to 2500 μg/mL.
- Results for the Relative Suspension Growth (%RSG) were as shown in the attached table.
- There was no evidence of any marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in any of the three exposure groups when compared to the concurrent vehicle control groups. Precipitate of the test item was observed at and above 39.06 μg/mL in all three of the exposure groups. Therefore, with no evidence of marked dose-related toxicity, the maximum dose level in the subsequent Mutagenicity Test was limited by the onset of test item precipitate.

MUTAGENICITY TEST
A summary of the results from the test is presented in Table 1 (attached).
- The results of the microtitre plate counts and their analysis are presented in Tables 2 to 10 (attached).
- There was no evidence of any marked toxicity following exposure to the test item in any of the three exposure groups, as indicated by the %RSG and RTG values (see Tables 3, 6, and 9, attached). There was also no evidence of any significant reductions in viability (%V) in any of the three exposure groups, indicating that residual toxicity had also not occurred (see Tables 3, 6, and 9, attached). Acceptable levels of toxicity were seen with the positive control substances (see Tables 3, 6, and 9, attached).
- Precipitate of the test item was observed at 40 and 80 μg/mL in the 4-hour exposure groups, and at 80 μg/mL in the 24-hour exposure group, at the end of the exposure periods. Therefore, as sufficient precipitating dose levels were observed (as recommended by the OECD 490 Guideline), the 80 μg/mL dose level in the 4-hour exposure groups was considered to be surplus to requirements and was not plated out for viability or 5-TFT resistance.
- 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 (see 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 10E-06 per viable cell at any of the dose levels (including the precipitating dose level), in any of the three exposure groups.
- The numbers of small and large colonies and their analysis are presented in Tables 4, 7, and 10 (attached).

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 in this assay.

Executive summary:

GUIDELINE

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 1.25, 2.5, 5, 10, 20, 40 µg/mL (4-hour without S9 and 4-hour with 2 % S9) and 2.5, 5, 10, 20, 40, 80 µg/mL (24-hour without S9).

 

RESULTS

The maximum dose level used in the Mutagenicity Test was limited by the onset of test item precipitate. 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.

 

CONCULSION

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)

Genetic toxicity in vivo

Description of key information

Negative results were obtained during investigation of in vitro gene mutation in bacteria (Ames test), in vitro cytogenicity in mammalian cells (chromosome aberration study) and in vitro gene mutation in mammalian cells (mouse lymphoma assay). As a result, and in accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 5.0; December 2016), the substance is not considered to be genotoxic and no further testing is required.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

The test 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.

 

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 atup to eight dose levels, in triplicate, both with and without the addition of a rat liverhomogenate metabolizing system (10% liver S9 in standard co-factors). The dose range forExperiment 1 was predetermined and was 1.5 to 5000μg/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 15 to 5000 μg/plate. Six test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the test item.

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. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test and consequently the same maximum dose level was used in the second mutation test. Similarly there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the second mutation test. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

 

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no toxicologically 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 withoutmetabolic activation (S9-mix) in Experiment 2. Small, statistically significant increases in TA1537 revertant colony frequency were observed in the second mutation test at 5000 μg/plate (absence of S9-mix only). This increase was considered to be of no biological relevance because there was no evidence of reproducibility or a dose-response relationship. Furthermore, the individual revertant counts at the statistically significant dose level were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.6 times the concurrent vehicle control.

 

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

 

Chromosome aberration test

Structural chromosomal aberrations were investigated in cultured mammalian cells in accordance with OECD Guidelines for Testing of Chemicals No 473 "In Vitro Mammalian Chromosome Aberration Test" adopted 26 September 2014 and the Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI) and Ministry of the Environment (MOE) Guidelines of 31 March 2011.

 

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at four dose levels, together with vehicle and positive controls. The study investigated three exposure conditions: 4 hours exposure in the presence of an induced rat liver homogenate metabolising system (S9) at a 2 % final concentration with cell harvest a 20 hour expression period; 4 hours exposure in the absence of metabolic activation (S9) with a 20 hour expression period; 24 hour exposure in the absence of metabolic activation. The dose levels used in the main experiment were selected using data from the preliminary toxicity test where results indicated that the maximum concentration should be limited on precipitate. The dose levels used in the main test were 0, 2.5, 5, 10, 15, 20, 40, 80 µg/L for 4(20) hour without S9; 0, 5, 10, 15, 20, 40, 80, 160 µg/L for 4(20) hour with S9 (2 %); 0, 2.5, 5, 10, 15, 20, 40, 80 µg/L for 24 hour without S9.

 

All vehicle (acetone) controls had frequencies of cells with aberration within the range expected for normal human lymphocytes. All the positive controls induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9 -mix were validated. The test item was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations using a dose range that included a concentration that was the lowest precipitating dose level.

 

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

Mouse lymphoma assay

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 B.17 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.

 

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 1.25, 2.5, 5, 10, 20, 40 µg/mL (4-hour without S9 and 4-hour with 2 % S9) and 2.5, 5, 10, 20, 40, 80 µg/mL (24-hour without S9).

 

The maximum dose level used in the Mutagenicity Test was limited by the onset of test item precipitate. 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.

 

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

Key in vitro tests demonstrated that the test material was non-mutagenic (Ames test and mouse lymphoma assay) and non-clastogenic (chromosome aberration test). In accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 5.0; December 2016), the substance is not considered to be genotoxic, in vivo testing is not required, and classification in accordance with Regulation (EC) No 1272/2008 does not apply.