Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: There wereno 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). The research material was considered to be non-mutagenic under the conditions of the test (OECD 471, EU Method B13/14, OPPTS 870.510 and relevant Japanese guidelines).

 

Chromosome aberration test: The research material was considered to be non-clastogenic to human lymphocytesin vitro.(OECD 473 and relevant 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 and OPPTS 870.5300).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 July 2016 to 19 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OCSPP harmonised guideline - Bacterial Reverse Mutation Test
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:
- Formulated concentrations were adjusted to allow for the stated water/impurity content (41 %) of the test item
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:
- Sterile distilled water
Negative solvent / vehicle controls:
yes
Remarks:
sterile distilled water
Untreated negative controls:
yes
Remarks:
untreated controls
Positive controls:
yes
Remarks:
without metabolic activation (2 µg/plate for WP2uvrA; 3 µg/plate for TA100 and 5 µg/plate for TA1535)
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
ENNG; direct acting compound; batch 67F-3700; 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 56-57-5; 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 613-13-8; 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 %; expiry date 12 October 2017; solvent DMSO
Details on test system and experimental conditions:
STUDY CONTROLS
- The solvent (vehicle) controls were performed in triplicate.
- The negative (untreated) controls were performed in triplicate to assess the spontaneous revertant colony rate.
- Sterility controls were performed for (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) the maximum dosing solution of the test item in the absence of S9-mix (singular test only).

MICROSOMAL ENZYME FRACTION
- The S9 microsomal fraction was pre-prepared using standardised in-house procedures.
- Lot number 05 June 2016 was used for the study.
- The certificate of S9 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 42228 09/16 and 42325 09/16).

BACTERIA
- Details of the five strains of bacteria used and their mutations are attached.
- All of the Salmonella strains were histidine dependent by virtue of a mutation through the histidine operon and were derived from S. typhimurium strain LT2 through mutations in the histidine locus. Additionally due to the “deep rough” (rfa) mutation they possessed a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through deletion of the uvrB bio gene, caused 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.
- In addition to a mutation in the tryptophan operon, the E. coli tester strain contained a uvrA DNA repair deficiency, which enhanced its sensitivity to some mutagenic compounds. This deficiency allowed 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.
- Bacteria used in the test were obtained from the University of California, Berkeley, on culture discs (04 August 1995) and the 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).
- Overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot number 1758279 10/20) and incubated at 37 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with tires 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 fully miscible in sterile distilled water at 50 mg/mL. Sterile distilled water was therefore selected as the vehicle.
- The test item was accurately weighed and approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer on the day of each experiment. Formulated concentrations were adjusted to allow for the stated water/impurity content (41 %) of the test item.
- 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 was reflected in the GLP compliance statement.

EXPERIMENT 1 – DOSE SELECTION
- Eight concentrations of the test item were assayed in triplicate against each tester strain (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate).
- The direct plate incorporation method was used.

EXPERIMENT 1 – WITHOUT METABOLIC ACTIVATION
- An aliquot (0.1 mL) of an 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 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).

EXPERIMENT 2 – DOSE SELECTION
- Based on the results of Experiment 1, the dose range chosen for Experiment 2 was 15 to 5000 µg/plate.
- Six test item dose levels were selected 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 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 due to the revertant colonies spreading slightly and distorting the actual plate count.

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.
- 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.
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.
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.
- 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 judgement about test item activity. Results of this type will be reported as equivocal.
Statistics:
- Statistical difference 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 (with and without metabolic activation) are given in Tables 2 and 3 for Experiment 1 (attached) and Tables 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 dose level of 5000 µg/plate.
- In the first mutation test, 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). 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 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 at any dose of test item, with and without metabolic activation (S9-mix).
- Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains at any dose level, with and without metabolic activation (S9-mix) in Experiment 2.
- The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range.
- All positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and 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 (30 May 2008) and the USA EPA OCSPP harmonised guideline – Bacterial Reverse Mutation Test.

 

METHODS

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 plus Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels. The investigation was performed in triplicate, with and without the addition of rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). 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 (sterile distilled water) 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, with and 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 the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial 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 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 item was considered to be non-mutagenic under the conditions of the test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 August 2016 to 18 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: chromosome aberration
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: 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 ºC with 5 % CO2
in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to
divide by the addition of phytohaemagglutinin (PHA).
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbitone/ß-Naphtha flavone induced S9 microsomal fraction (see Appendix 2, attached)
Test concentrations with justification for top dose:
PRELIMINARY TEST
0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000, 2000 μg/mL

MAIN TEST
- 4 (20) hour without S9: 0, 62.5, 125, 250, 500, 1000, 2000 μg/mL
- 4 (20) hour with S9 (2 %): 62.5, 125, 250, 500, 1000, 2000 μg/mL
- 24 hour without S9: 62.5, 125, 250, 500, 1000, 2000 μg/mL
Vehicle / solvent:
Minimal Essential Medium (MEM)
Negative solvent / vehicle controls:
yes
Remarks:
MEM
Remarks:
Batch RNBF2732 (Preliminary Toxicity Test and Main Test)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
mitomycin C
Remarks:
MMC (Sigma Aldrich; CAS 50-07-7; Batch SLBM6258V; Purity assumed to be 100 %; Expiry date 01 May 2019; Solvent Minimal Essential Medium)
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
cyclophosphamide
Remarks:
CP (Acros Organics; CAS 6055-19-2; Batch A0355340; Purity assumed to be 97 %; Expiry date 01 January 2020; Solvent DMSO)
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 been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.
- The donor was male, aged 30 years, for the preliminary toxicity test and male, aged 28 years, for the main experiment.
- 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
- The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/βNF S9 10/04/016 was used in this study. A copy of the S9 Certificate of Efficacy is presented in Appendix 2 (attached).
- The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

TEST ITEM PREPARATION AND ANALYSIS
- The molecular weight of the test item was given as 693.9, therefore, the maximum dose level was 2000 μg/mL, which was calculated to be equivalent to the maximum recommended dose level.
- The purity of the test item was 59 % and was accounted for in the test item formulations. The test item was soluble in tissue culture media (MEM) at 50 mg/mL in solubility checks performed in-house.
- Prior to each experiment, the test item was accurately weighed, formulated in tissue culture media (MEM) and appropriate serial dilutions prepared.
- There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).
- The pH and osmolality readings are presented in the table below.
- The test item was formulated within two hours of it being applied to the test system; the test
item formulations were assumed to be stable.
- No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This was 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.
- Each culture dispensed into plastic flasks contained MEM 10 % FBS (8.05 mL); Li-heparin (0.1 mL); phytohaemagglutinin (0.1 mL); heparinised whole blood (0.75 mL).

4-HOUR EXPOSURE WITH METABOLIC ACTIVATION (S9)
- After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment.
- After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 °C in 5 % CO2 in humidified air.

4-HOUR EXPOSURE WITHOUT METABOLIC ACTIVATION (S9)
- After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
- After 4 hours at approximately 37 ºC, 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 (S9)
- As the exposure was continuous the cultures were established, at a nominal volume of 9.0 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 °C, 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 hours 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 hours 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.
- The dose range of test item used was 7.81 to 2000 μg/mL.
- 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 evaluation 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 EXPERIEMENT
- 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. The dose range of test item used was 62.5 to 2000 μg/mL.
(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. The dose range of test item used was 62.5 to 2000 μg/mL.
(iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 62.5 to 2000 μg/mL.

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, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl.
- After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCI cell 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 resuspension
in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

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

QUALITATIVE SLIDE ASSESSMENT
- The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. 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 recorded and 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 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors. The current historical range is shown in Appendix 1 (attached).

CRITERIA FOR DETERMINING THE STUDY CONCLUSION
- Providing that all of the acceptability criteria are fulfilled, a test item can 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 should be 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 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 the laboratory 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.
- When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
- Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells.

MAJOR COMPUTERISED SYSTEMS
- Data analysis was performed using an in-house developed program.
- Delta Building Monitoring System.
Evaluation criteria:
DATA EVALUATION
- The following criteria were used to determine a valid assay:
(i) The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range. The level of spontaneous background aberrations was slightly elevated above the normal range and the experiment still considered valid.
(ii) All the positive control chemicals induced a positive response (p ≤ 0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
(iii) The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
(iv) The required number of cells and concentrations were analysed.
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 is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
PRELIMINARY TOXICITY TEST
- The dose range for the Preliminary Toxicity Test was 7.81 to 2000 μg/mL, the maximum recommended dose level.
- No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure period in all three exposure groups.
- Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 2000 μg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 2000 μg/mL. The mitotic index data are presented in Table 1 (attached).
- The selection of the maximum dose level for the Main Experiment was based on the maximum recommended dose level and was 2000 μg/mL for the 4(20)-hour exposure groups and for the continuous exposure group.

CHROMOSOME ABERRATION TEST – MAIN EXPERIMENT
- The dose levels of the controls and the test item are given in the table below.
- The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 2000 μg/mL in all three exposure groups.
- The mitotic index data for the Main Experiment are given in Table 2 and Table 3 (attached). They confirm the qualitative observations in that a slight dose-related inhibition of mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, 10% mitotic inhibition was achieved at 2000 μg/mL. In the presence of S9, 23% mitotic inhibition was achieved at 2000 μg/mL. An inhibition of mitotic index of 40% was noted at 2000 μg/mL in the 24-hour continuous exposure group. The maximum dose level selected for metaphase analysis was the maximum recommended dose level (2000 μg/mL).
- The chromosome aberration data are given in Table 4, Table 5 and Table 6 (attached). The assay was considered valid as it met all of the following criteria: The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range. All the positive control chemicals induced a demonstrable positive response (p ≤ 0.01) and
confirmed the validity and sensitivity of the assay and the integrity of the S9-mix. The study was 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 were analysed. 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.
- The polyploid cell frequency data are given in Table 7 (attached). 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.

DOSE LEVELS OF CONTROLS AND TEST ITEM

Group

Final concentration of test item (µg/mL)

4(20)-hour without S9

0*, 62.5, 125, 250*, 500*, 1000*, 2000*, MMC 0.2*

4(20)-hour with S9 (2 %)

0*, 62.5, 125, 250*, 500*, 1000*, 2000*, CP 2*

24-hour without S9

0*, 62.5, 125, 250*, 500*, 1000*, 2000*, MMC 0.05*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

Conclusions:
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 26th September 2014 and 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.

 

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. 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 a 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 selected for the Main Test were 0, 62.5, 125, 250, 500, 1000 and 2000 µg/mL for 4(20)-hour without S9), 4(20)-hour with S9 (2%) and 24-hour without S9.

 

RESULTS

All vehicle (MEM) 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. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the maximum recommended 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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 August 2016 to 19 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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: mouse lymphoma assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
3.7.2c
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:
125, 250, 500, 1000, 1500 and 2000 µg/mL for 4-hour without S9, 4-hour with S9 (2 %) and 24-hour without S9.
Vehicle / solvent:
R0 medium
Negative solvent / vehicle controls:
yes
Remarks:
R0 medium
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
ethylmethanesulphonate
Remarks:
see details of control preparation (below)
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
cyclophosphamide
Remarks:
see details of control preparation (below)
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), are 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.
- The 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. This 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 test item, the material was accurately weighed and formulated in R0 medium prior to serial dilutions being prepared. The molecular weight of the test item was 693.9, therefore the maximum proposed dose level in the solubility test was set at 2000 μg/mL, the maximum recommended dose level, and a correction for the stated water / impurity content (41%) of the test item of the test item was applied.
- 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 shown 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 was an exception with regard to GLP and was reflected in the GLP compliance statement.

CONTROL PREPARATION
- Vehicle and positive controls were used in parallel with the test item. Solvent (R0 medium) exposure groups were used as the vehicle controls.
- Ethylmethanesulphonate (EMS) (Sigma batch BCBQ0451V, purity 100%, expiry 12.02.17) at 400 μg/mL and 150 μg/mL, respectively, was used as the positive control in the 4-hour and 24-hour exposure groups in the absence of metabolic activation.
- Cyclophosphamide (Acros Organics batch A0355340, purity 97%, Expiry 24.11.17) at 1.5 μg/mL was used as the positive control in the presence of metabolic activation. The positive controls were formulated in DMSO.

PRELIMINARY TOXICITY TEST
- A preliminary toxicity test was performed on cell cultures at 5 x 105 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 7.81 to 2000 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 10E05 cells/mL, unless the mean cell count was less than 3 x 10E05 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 106 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 six dose levels of the test item (125 to 2000 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, 2 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 visualisation 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 using the equation RCE = %V / mean solvent control %V
- The RTG was then calculated for each culture using the equation RTG = (RCE * RSG) / 100

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.

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 are 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 to 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) The upper test item concentrations will be 10mM, 2 mg/mL or 2 μL/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
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
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 7.81 to 2000 μg/mL. The 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 the 4-hour exposure groups in both the absence and presence of metabolic activation when compared to the concurrent vehicle control groups. However, marked reductions were observed in the 24-hour exposure group in the absence of metabolic activation when compared to the vehicle control group. No precipitate of the test item was observed at any of the dose levels in any of the three exposure groups. Therefore, the maximum dose level in the subsequent Mutagenicity Test was the maximum recommended dose level of 2000 μg/mL.

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).
- As was seen previously, there was no evidence of any marked toxicity following exposure to the test item in either of the 4-hour exposure groups, and marked toxicity in the 24-hour exposure group, as indicated by the %RSG and RTG values (see Tables 3, 6, and 9, attached). There was no evidence of any significant reductions in viability (%V) in any of the three exposure groups, indicating that residual toxicity had not occurred (see Tables 3, 6, and 9, attached). Based on the RTG and / or %RSG values observed, optimum levels of toxicity were considered to have been achieved in the 24-hour exposure group (see Table 9, attached). Acceptable levels of toxicity were seen with the positive control substances (see Tables 3, 6, and 9, attached).
- No precipitate of the test item was observed at any of the dose levels in any of the three exposure groups at the end of the exposure periods.
- 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 (Tables 3, 6, and 9).
- 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 maximum recommended 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 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.

 

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 six dose levels in duplicate, together with vehicle (R0 medium), 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 125, 250, 500, 1000, 1500 and 2000 µg/mL for 4-hour without S9, 4-hour with S9 (2 %) and 24-hour without S9.

 

RESULTS

The maximum dose level used in the Mutagenicity Test was the maximum recommended dose level of 2000 μg/mL. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolising 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

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay

In accordance with OECD 471, EU Method B13/14 and other applicable guidelines, the key study involved Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 plus Escherichia coli strain WP2uvrA treated with the test item using the Ames plate incorporation method at up to eight dose levels. The investigation was performed in triplicate, with and without the addition of rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). 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 (sterile distilled water) 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, with and 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 the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial 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 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 test item was considered to be non-mutagenic under the conditions of the test.

Chromosome aberration test

Structural chromosomal aberrations were investigated in a key study using cultured mammalian cells in accordance with OECD Guidelines for Testing of Chemicals No. 473 "In Vitro Mammalian Chromosome Aberration Test" adopted 26th September 2014 and 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.

 

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. 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 a 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 selected for the Main Test were 0, 62.5, 125, 250, 500, 1000 and 2000 µg/mL for 4(20)-hour without S9), 4(20)-hour with S9 (2%) and 24-hour without S9.

 

All vehicle (MEM) 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. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the maximum recommended 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 key 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 Vitro Mammalian 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 six dose levels in duplicate, together with vehicle (R0 medium), 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 125, 250, 500, 1000, 1500 and 2000 µg/mL for 4-hour without S9, 4-hour with S9 (2 %) and 24-hour without S9.

 

The maximum dose level used in the Mutagenicity Test was the maximum recommended dose level of 2000 μg/mL. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolising 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.

In vivo

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 6.0; July 2017), the substance is not considered to be genotoxic and no further testing is required.

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 6.0; July 2017), 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.