Reaction products of dimethyl phosphonate with 2-alkyl-2-alkylpropanediol and alkanediol

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, OPPTS 870.5100 and relevant Japanese guidelines).

 

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 enzymemetabolising system. The test item was therefore considered to be non-clastogenic to human lymphocytes in vitro (OECD 473 and relevant Japanese guidelines).

Mouse lymphoma assay: The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test with or without metabolic activation. The test item was therefore considered to be non-mutagenic (OECD 490, EU Method B.17, OPPTS 870.5300 and relevant 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:

04 January 2017 to 02 February 2017

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:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

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:

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:

without

Metabolic activation system:

Phenobarbital / ß-Naphtha flavone induced 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: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate

Vehicle / solvent:

Dimethyl sulphoxide (DMSO)

Untreated negative controls:

yes

Remarks:

untreated controls

Negative solvent / vehicle controls:

yes

Positive control substance:

other: dimethyl sulphoxide (DMSO)

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

ENNG (CAS number 4245-77-6; Batch 67F-3700; Purity not available; Expiry date 18 September 2017; Solvent DMSO)

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

9-aminoacridine

Remarks:

9AA (CAS number 90-45-9; Batch S32398-438; Purity 99.9 %; Expiry date 01 October 2017; Solvent DMSO)

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

4NQO (CAS number 56-57-5; Batch 030M1206; Purity 100 %; Expiry date 08 October 2017; Solvent DMSO)

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA (CAS number 613-13-8; Batch STBB1901M9; Purity 97.5 %; Expiry date 08 October 2017; Solvent DMSO)

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

benzo(a)pyrene

Remarks:

BP (CAS number 50-32-8; Batch 090M1400V; Purity 96 %; Expiry date 12 October 2017; Solvent DMSO)

Details on test system and experimental conditions:

STUDY CONTROLS
- The solvent (vehicle) control used was dimethyl sulphoxide. The negative (untreated) controls were performed to assess the spontaneous revertant colony rate. The 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. The positive controls were performed in triplicate.

STERILITY CONTROLS
- Top agar and histidine/biotin or tryptophan in the absence of S9-mix (in triplicate).
- Top agar and histidine/biotin or tryptophan in the presence of S9-mix (in triplicate).
- The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).

MICROSOMAL ENZYME FRACTION
- The S9 Microsomal fractions were pre-prepared using standardised in-house procedures (outside the confines of this study).
- Lot number 22 September 2016 was used in this study.
- A copy of the S9 Certificate of Efficacy are 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.
- The S9 mix contained 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); sterile distilled water (14.5 mL).
- A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar 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 each experiment.
- 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 was added to each 100 mL of top agar.
- Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (Lot numbers 43372 02/17 and 43609 03/17).

BACTERIA
- The five strains of bacteria used are shown in the table below together with their mutations.
- All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine operon and are derived from S. typhimurium strain L T2 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 TAI 00~ the R-factor plasmid pKMIOI 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 auvrA-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).
- The bacteria used in the test were obtained from the University of California, Berkeley, on culture discs, on 04 August 1995 or from the British Industrial Biological Research Association, on a nutrient agar plate, on 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 number 1865318 05/21) 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
- In solubility checks performed in-house the test item was noted to be immiscible in sterile distilled water at 50 mg/mL but fully miscible in dimethyl sulphoxide at the same concentration. Dimethyl sulphoxide was selected as the vehicle.
- The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment. No correction was required for test item purity. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
- 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.

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

EXPERIMENT 1 - WITHOUT METABOLIC ACTIVATION
- An aliquot (0.1 mL) of the appropriate concentration of test item or solvent vehicle or 0.1 mL of appropriate positive control was added to 2 mL of molten, trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate.
- Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

EXPERIMENT 1 - WITH METABOLIC ACTIVATION
- The procedure was the same as described previously except that following addition of the 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).
- A single manual count was required due to revertant colonies spreading slightly, thus distorting the actual plate count.

DOSE SELECTION FOR EXPERIMENT 2
- The dose range used for Experiment 2 was determined by the results of Experiment 1 and was the same as the first mutation test (1.5 to 5000 μg/plate).
- Eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item.

EXPERIMENT 2 - WITHOUT METABOLIC ACTIVATION
- The procedure was the same as described previously.

EXPERIMENT 2 - WITH METABOLIC ACTIVATION
- The procedure was the same as described previously.

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).

ACCEPTANCE CRITERIA
- The reverse mutation assay may be considered valid if the following criteria are met:
(i) All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
(ii) 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 TA 1535 (7 to 40); TA100 (60 to 200); TA1537 (2 to 30); TA98 (8 to 60); WP2uvrA (10 to 60). Combined historical negative and solvent control ranges for 2012 and 2013 are presented in Appendix 1 (attached).
(iii) All tester strain cultures should be in the range of 0.9 to 9 x 10E09 bacteria per mL.
(iv) Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix. All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation. The historical ranges of the positive control reference items for 2014 and 2015 are presented in Appendix 1 (attached).
(v) There should be a minimum of four non-toxic test item dose levels.
(vi) There should be no evidence of excessive contamination.

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

Evaluation criteria:

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:
(i) A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
(ii) A reproducible increase at one or more concentrations.
(iii) Biological relevance against in-house historical control ranges.
(iv) Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
(v) Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by · an out-of-historical range response (Cariello and Piegorsch, 1996).
- A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

STATISTICAL ANALYSIS
- 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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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 are 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 Table 2 and Table 3 (attached) for Experiment 1 and Table 4 and Table 5 (attached) for Experiment 2.
- 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. The test item induced a visible reduction in the growth of the bacterial background lawns of TA100, in the absence of S9-mix, from 1500 μg/plate and at 5000 μg/plate to TA1535 (absence of S9-mix) and TA100 and TA1535 in the presence of S9-mix. No toxicity was noted to any of the remaining bacterial tester strains in either the absence or presence of S9-mix. Consequently the same maximum dose level was used in the second mutation test. A similar toxic response was noted in Experiment 2 with weakened bacterial background lawns noted to TA100 and TA1535 at 5000 μg/plate in both the presence and absence of S9-mix. Once again, no toxicity was noted to TA98, TA1537 and WP2uvrA at any test item dose level in either the absence or presence of S9-mix. 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 vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated

Remarks on result:

other: see information on cytotoxicity below

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 at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10 % liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was not amended following the results of Experiment 1 and was 1.5 to 5000 μg/plate. Eight 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 (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

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

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 January 2017 to 17 March 2017

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 % fetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital / ß-Naphtha flavone induced S9 mix

Test concentrations with justification for top dose:

MAIN STUDY
- 4(20)-hour without S9: 10, 20, 40, 80, 100, 120, 160 μg/mL
- 4(20)-hour with S9 (2%): 20, 40, 80, 160, 200, 240, 320 μg/mL
- 24-hour without S9: 20, 40, 80, 160, 200, 240, 320 μg/mL

Vehicle / solvent:

Dimethyl sulphoxide

Negative solvent / vehicle controls:

yes

Positive control substance:

other: Dimethyl sulphoxide

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

mitomycin C

Remarks:

MMC (Sigma Aldrich; CAS number 50-07-7; Batch SLBM6528V; Purity treated as 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 number 50-18-0; Batch A0355340; Purity 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.
- 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.
- The donor for the preliminary test was male (aged 25 years) and the donor for the main experiment was male (aged 27 years).

MICROSOMAL ENZYME FRACTION
- Lot No 22/09/16 PB/βNF S9 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 was tested for its capability to activate known mutagens in the Ames test and a Certificate of Efficacy is presented in Appendix 5.
- The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (10 to 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% in the Cell Growth Inhibition Test and Main Experiment.

TEST ITEM PREPARATION AND ANALYSIS
- The test item was considered to be a UVCB and therefore the maximum recommended dose level was 5000 μg/mL. A purity correction was not required.
- The test item was insoluble in Minimal Essential Medium at 50 mg/mL but was soluble in dimethyl sulphoxide (DMSO) at 500 mg/mL in solubility checks performed in-house.
- Prior to each experiment, the test item was accurately weighed, formulated in DMSO 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 osmolarity readings are shown 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 for this duration. 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 is an exception with regard to GLP and has been 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 °C, 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 0.1 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 Cell Growth Inhibition Test (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 °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 resuspended in the required volume of fresh MEM (including serum) and dosed with 0.1 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, 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)
- For the 24-hour exposure in the absence of S9, the exposure was continuous. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.1 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 Cell Growth Inhibition 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 19.53 to 5000 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 10, 20, 40, 80, 100, 120 and 160 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 20, 40, 80, 160, 200, 240 and 320 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 20, 40, 80, 160, 200, 240, 320 μg/mL.

CELL HARVEST
- Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two and a half 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 KCl 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 re-suspension 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 4, attached). 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. A dose-related increase in endoreduplicated cells are reported separately. 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 4 (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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
- The dose range for the Cell Growth Inhibition Test was 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/mL. The maximum dose was the maximum recommended dose level.
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of exposure, at and above 1250 μg/mL, in the 4(20)-hour exposure groups and at and above 2500 μg/mL in the continuous exposure group.
- Haemolysis was observed following exposure to the test item at and above 312.5 μg/mL in all three exposure groups. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
- Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 78.13 μg/mL in the 4(20)-hour exposure in the absence of metabolic activation (S9). The maximum dose level with metaphases present in the 4(20)-hour exposure in the presence of S9 and in the 24-hour exposure group was 156.25 μg/mL. The results of the mitotic index of the Cell Growth Inhibition Test are presented in sections 5(2) and 6(2) of Appendix 1. The test item induced marked evidence of toxicity in all three exposure groups and demonstrated a very steep toxicity curve.
- The selection of the maximum dose level for the Main Experiment was based on toxicity and was 160 μg/mL for the 4(20)-hour exposure group in the absence of S9 and 320 μg/mL for the 4(20)-hour exposure group in the presence of S9 and for the 24-hour 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 Cell Growth Inhibition Test and that there were metaphases suitable for scoring present up to 120 μg/mL and 200μg/mL in the 4(20)-hour exposure groups in the absence and presence of S9, respectively. In the 24-hour continuous exposure group the maximum dose level of the test item with metaphases suitable for scoring was 240 μg/mL.
- Precipitate observations were made in the blood cultures at the end of exposure and no precipitate was observed. Haemolysis was observed at the end of exposure at 160 μg/mL in the 4(20)-hour exposure in the absence of S9, at and above 80 μg/mL in the 4(20)-hour exposure in the presence of S9 and at 320 μg/mL in the 24-hour exposure group. These data are presented in Appendix 1, section 5(2) and 6(2).
- The results of the mitotic indices (MI) from the cultures after their respective exposures are presented as cell growth indices in Form 1, Form 2, and Form 3 in Appendix 2. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, optimum toxicity with 49% mitotic inhibition was achieved at 100 μg/mL. The dose level of 120 μg/mL in this exposure group demonstrated 73% mitotic inhibition and was considered too toxic for metaphase analysis. In the presence of S9, optimum toxicity was achieved at 160 μg/mL with 50% inhibition of mitotic index and the dose level of 200 μg/mL was too toxic for scoring with 93% mitotic inhibition. The 24-hour exposure group demonstrated a dose related inhibition of mitotic index with 39% and 75% mitotic inhibition at 200 μg/mL and 240 μg/mL, respectively.
- The maximum dose level selected for metaphase analysis was based on toxicity and was 100 μg/mL for the 4(20)-hour exposure group in the absence of S9 and 160 μg/mL for the 4(20)-hour exposure group in the presence of S9, where optimum toxicity was achieved. The maximum dose level selected for scoring in the 24-hour exposure group was 240 μg/mL where the toxicity was greater than optimum but it was an intermediate dose level in a steep toxicity curve.
- The chromosome aberration data are given in Form 1, Form 2 and Form 3, Appendix 2 (attached). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range.
- All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.
- The 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 Figure 2, Figure 3 and 4 and Forms 1, 2 and 3 (attached). The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the three exposure groups.

DOSE LEVELS OF CONTROLS AND TEST ITEM

Group	Final concentration of test item (µg/mL)
4(20)-hour without S9	0*, 10, 20, 40*, 80*, 100*, 120, 160, MMC 0.4*
4(20)-hour with S9 (2 %)	0*, 20, 40*, 80*, 160*, 200, 240, 320, CP 2*
24-hour without S9	0*, 20, 40, 80*, 160*, 200*, 240*, 320, MMC 0.2*

* = 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 29 July 2016 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 up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after 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 used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the Main Test were 10, 20, 40, 80, 100, 120, 160 μg/mL (4(20)-hour without S9 exposure group), 20, 40, 80, 160, 200, 240, 320 μg/mL (4(20)-hour with S9 (2%) exposure group) and 20, 40, 80, 160, 200, 240, 320 μg/mL (24-hour without S9 exposure group).

 

RESULTS

All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item demonstrated marked toxicity in all three exposure groups and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that induced approximately 50% mitotic inhibition or greater.

 

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 January 2017 to 07 February 2017

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:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

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

Test concentrations with justification for top dose:

The dose levels plated for viability and expression of mutant colonies were 10, 20, 40, 60, 80, 100 μg/mL (4-hour without S9 group), 40, 80, 100, 120, 140, 160 μg/mL (4-hour with S9 (2%) group) and 40, 80, 100, 120, 160, 200 μg/mL (24-hour without S9 group).

Vehicle / solvent:

Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Positive control substance:

other: Dimethylsulfoxide

Remarks:

DMSO (Fluka; Batch SZBG083OV; Expiry date 08 March 2019; Purity ≥ 99.9 %)

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Remarks:

EMS (Sigma; Batch BCBQ0451V; Expiry date 04 November 2021; Purity treated as 100 %; Solvent DMSO)

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

CP (Acros Organics; Batch A0355340; Expiry date 01 January 2020; Purity 97 %; Solvent DMSO)

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 sub-cultured 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 22/09/16 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 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%.

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 on the corresponding Human Lymphocyte Chromosome Aberration test KB55NS, the test item was accurately weighed and formulated in DMSO prior to serial dilutions being prepared.
- The test item was a UVCB compound and the maximum recommended dose level was therefore set at 5000 μg/mL with no correction for purity. 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 are presented in the table below.
- No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This was 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 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 was set at 19.53 to 5000 μg/mL in all three exposure groups. Following the exposure period, the cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 x 105 cells/mL.
- 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 105 cells/mL. 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 treatment toxicity, and a comparison of each treatment 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) Maximum recommended dose level, 5000 μg/mL or 10 mM, whichever is the lowest concentration.
ii) The presence of precipitate regardless of where test item-induced toxicity was observed.
iii) Test item-induced toxicity, where the maximum dose level used should produce 10 to 20% survival (the maximum level of toxicity required). 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 8 dose levels of the test item (10 to 160 μg/mL for both 4-hour exposure groups and 10 to 200 μg/mL in the 24-hour exposure group), vehicle and positive controls. 2 mL of S9-mix if required, 0.2 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) were added to each universal.
- 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 are 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.

Evaluation criteria:

See below

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 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 19.53 to 5000 μg/mL. The results for the Relative Suspension Growth (%RSG) were as shown in the attached table.
- In the 4-hour exposures, both in the absence and presence of metabolic activation (S9), there was no evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. In the 24-hour exposure there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. A precipitate of the test item was observed at and above 625 μg/mL in the 4-hour –S9 exposure group and 1250 μg/mL and above in the 4-hour +S9 and 24-S9 exposure groups. In the subsequent mutagenicity experiments the maximum dose level was limited by test item induced toxicity.

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 evidence of marked toxicity following exposure to the test item in all three of the exposure groups, as indicated by the %RSG and RTG values (see Tables 3, 6, and 9, attached).
- There was evidence of reductions in viability (%V) in the 4-hour +S9 dose group, therefore indicating that residual toxicity had occurred in this dose group only (see Table 6, attached).
- Very near to optimum levels of toxicity were considered to have been achieved in all three exposure groups. In the 4-hour exposure in the absence of metabolic activation, the 120 and 160 μg/mL dose levels were not plated out for 5-TFT resistance and viability due to excessive toxicity.
- In the 4-hour exposure in the presence of metabolic activation, at 160 μg/mL, and in the 24-hour exposure in the absence of metabolic activation, at 200 μg/mL although these dose levels were plated out for 5-TFT resistance and viability, were later excluded from analysis due to excessive toxicity. Acceptable levels of toxicity were seen with both positive control substances (see Tables 3, 6 and 9, attached).
- 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 increases in the mutant frequency x 10E-06 per viable cell in either of the three exposure groups. The GEF value of the test item dose levels were not exceeded in any of the three exposure groups, including dose levels beyond the acceptable level of toxicity. No precipitate was observed throughout. 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 29 July 2016, 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 8 dose levels in duplicate, together with vehicle (DMSO), 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 test item exhibited marked toxicity in all three exposure groups. Precipitate of the test item was also observed in all three of the exposure groups. The dose levels plated for viability and expression of mutant colonies were 10, 20, 40, 60, 80, 100 μg/mL for 4-hour without S9, 40, 80, 100, 120, 140, 160 μg/mL for 4-hour with S9 (2%) and 40, 80, 100, 120, 160, 200 μg/mL for 24-hour without S9.

 

RESULTS

The maximum dose level used was limited by test item induced toxicity. No precipitate of the test item was observed throughout the main test. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. Very near to optimum levels of toxicity were considered to have been achieved in all three exposure groups. 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

Mouse micronucleus test: The test item was considered to be non-genotoxic (OECD 474, EU Method B.12 and OPPTS 870.5395).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 April 2017 to 27 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

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: damage to chromosomes and/or aneuploidy

Species:

mouse

Strain:

other: Hsd: ICR (CD-1)

Remarks:

albino

Sex:

male

Details on test animals or test system and environmental conditions:

ANIMALS AND ANIMAL HUSBANDRY
- Sufficient albino Hsd: ICR (CD-1) strain mice were obtained from Harlan Laboratories UK Ltd, Oxon, UK.
- At the start of the main test, the mice weighed 23.5 to 30.9 g and were approximately six to ten weeks old.
- After a minimum acclimatisation period of five days, the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.
- The animals were housed in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding.
- Free access to mains drinking water and food (Harlan Teklad 2014C Global Certified Rodent diet supplied by Harlan Laboratories Ltd, Oxon, UK) was allowed throughout the study. Representative analyses of food and water quality are retained in the laboratory archive.
- Temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70 % respectively.
- The rate of air exchange was approximately 15 changes per hour.
- Lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Route of administration:

oral: gavage

Vehicle:

VEHICLE
- Identification: Arachis oil
- Label number: ZZZ08613
- Envigo serial number: V-6498
- Purity: Treated as 100 %
- Expiry date: 12 May 2018
- Storage conditions: Room temperature

Details on exposure:

PURPOSE OF THE TEST
- The micronucleus test is a mammalian in vivo test that detects damage to the chromosomes induced by chemicals. In addition, numerical changes due to chromosome loss during cell division can be detected by the test. The results are believed to be of value in predicting the mutagenic potential of the test item to man. The test system was chosen because the mouse has been shown to be a suitable model for this type of study and is recommended in the test method.
- In mitotic cells in which chromosome damage has been caused by the test item or its metabolites, fragments (centric or acentric) or whole chromosomes tend to lag behind in the anaphase stage of cell division. After telophase a large proportion of the fragments are not included in the nuclei of the daughter cells and hence form a single or multiple micronuclei (Howell-Jolly bodies) in the cytoplasm of these cells. These micronuclei are seen in a wide variety of cell types but erythrocytes are chosen since micronuclei are easily detected in these cells.
- A few hours after the last mitosis is completed, erythrocytes expel their nuclei. Immature erythrocytes, less than 24 hours old, stain blue with May-Grünwald/Giemsa due to the presence of minute fragments of nuclear material in the cytoplasm. This material is mainly ribonucleic acid (RNA), which gradually disappears so that more mature erythrocytes (normochromatic erythrocytes) stain pink with May-Grünwald/Giemsa. The immature blue-staining cells are known as polychromatic erythrocytes and mauve-stained micronuclei are easily detected in this cell type. If scoring is restricted to polychromatic erythrocytes, all the chromosomal damage detected will have been caused during the final cell cycle of the nucleated precursor cells. Thus by examining polychromatic cells at various periods after administration, the effect of the test item over the previous 30 hours can be monitored.
- Any toxic effects of the test item on the immature nucleated cells may lead to a reduction in cell division and cell death. This in turn leads to a reduction in cell volume and, to compensate for this, peripheral blood is shunted into the bone marrow. If the ratio of polychromatic to normochromatic erythrocytes is scored and found to be significantly lower than the control value, this is taken to be indicative of cytotoxicity.

PREPARATION OF TEST ITEM
- When required, the test item was freshly prepared as a solution at the appropriate concentration in arachis oil.
- 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 and it was assumed the formulation was stable over that duration. This is an exception with regard to GLP and was reflected in the GLP compliance statement.

PREPARATION OF POSITIVE CONTROL ITEM
- When required, the positive control item was freshly prepared as a solution at the appropriate concentration in distilled water (Laboratoire Aguettant batch number 3012436).

RANGE-FINDING TOXICITY TEST
- A range-finding toxicity test was performed to determine a suitable dose level and route of administration for the main test.
- The dose level selected should ideally be the maximum tolerated dose level, or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.
- The range-finding test was also used to determine if the main test should be performed using both sexes or males only.
- Bone marrow samples were taken from the range-finding animals. Slides were then prepared and qualitatively assessed to ensure that any bone marrow toxicity observed was within acceptable limits for the main test.
- Groups of mice were dosed as shown in the table below.
- All animals were dosed once only at the appropriate dose level.
- Dosing was by gavage using a metal cannula or with a hypodermic needle attached to a graduated syringe.
- The volume administered to each animal was calculated according to its bodyweight at the time of dosing.
- Animals were observed one hour after dosing and subsequently once daily for two days.
- Any deaths and evidence of overt toxicity were recorded at each observation.
- No necropsies were performed.

MICRONUCLEUS TEST
- Groups of seven mice were dosed with the test item once only via the oral route. Doses received were 2000, 1000 or 500 mg/kg.
- One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test item at 2000 mg/kg was killed after 48 hours.
- In addition, two further groups of mice were included in the study; one group (five mice) was dosed via the oral route with the vehicle alone (arachis oil) and second group (five mice) was dosed orally with cyclophosphamide, which is a positive control item known to produce micronuclei under the conditions of the test.
- The vehicle controls and positive control group animals were killed 24 hours after dosing.
- Experimental design is summarised in the table below.
- All animals were observed one hour after dosing and then once daily and immediately prior to termination.

Duration of treatment / exposure:

24 or 48 hours

Frequency of treatment:

Single dose

Post exposure period:

Not applicable

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Seven

Control animals:

yes, concurrent vehicle

Positive control(s):

POSITIVE CONTROL
- Identification: Cyclophosphamide monohydrate
- Supplier: Acros Organics
- Lot number: A0373263
- Envigo serial number: R-6737
- Purity: 97 %
- Expiry date: 22 February 2019
- Storage conditions: Approximately 4 °C in the dark

Tissues and cell types examined:

Both femurs were dissected from each animal immediately following sacrifice.

Details of tissue and slide preparation:

SLIDE PREPARATION
- Immediately following termination (24 or 48 hours after dosing), both femurs were dissected from each animal, aspirated with foetal bovine serum and bone marrow smears prepared following centrifugation and re-suspension.
- The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa, allowed to air-dry and a cover slip was applied using mounting medium.

SLIDE EVALUATION
- Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification.
- The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored.
- Micronuclei are normally circular in shape although, occasionally, they may be oval or half-moon shaped. The micronuclei have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted. These cells were also scored for incidence of micronuclei.
- The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.

Evaluation criteria:

DATA EVALUATION
- Comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group.
- A positive mutagenic response is demonstrated when a statistically significant, dose-responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24 or 48-hour kill times when compared to the vehicle control group.
- If these criteria were not fulfilled, then the test item was considered to be non-genotoxic under the conditions of the test.
- A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the vehicle control group.

Statistics:

STATISTICAL ANALYSIS
- All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989).
- The data was analysed following a transformation using Student's t-test (two tailed).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING TOXICITY TEST
- Mortality data are summarised in the table below.
- Clinical signs were not observed in any of the animals dosed at 1000 mg/kg, or the maximum recommended dose of 2000 mg/kg. Qualitative analysis of the bone marrow slides indicated that there was evidence of very modest toxicity to the bone marrow at 2000 mg/kg.
- The micronucleus test was therefore conducted using the oral route in groups of seven mice (males) at the maximum recommended dose of 2000 mg/kg, with 1000 and 500 mg/kg as the two lower dose levels. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice.

MICRONUCLEUS TEST
- There were no premature deaths or clinical signs observed in any of the dose groups in the main test.

EVALUATION OF BONE MARROW SLIDES
- A summary of results of the micronucleus test is given in Table 1 (attached).
- Historical control data for studies performed between 2014 and 2017 is summarised in Appendix 1 (attached).
- Individual and group mean data are presented in Tables 2 to 7 (attached).
- There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test item groups when compared to the vehicle control group. However, it should be noted that the vehicle control PCE/NCE ratio was quite low.
- There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test item dose groups when compared to the vehicle control group.
- The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

MORTALITY DURING RANGE-FINDING TOXICITY TEST

Dose level (mg/kg)	Sex	Number of animals treated	Route	Deaths on day 0	Deaths on day 1	Deaths on day 2
1000	Male	1	Oral	0	0	0
1000	Female	1	Oral	0	0	0
2000	Male	1	Oral	0	0	0
2000	Female	1	Oral	0	0	0
2000	Male	2	Oral	0	0	0

Conclusions:

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

Executive summary:

GUIDELINE

The study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 474 “Mammalian Erythrocyte Micronucleus Test” (adopted 29 July 2016), Method B.12 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA (TSCA) OPPTS 870.5395, EPA 712-C-98-226, August 1998 guidelines, and be acceptable to the Japanese METI/MHLW/MAFF guidelines for testing of new chemical substances.

 

METHODS

A range-finding test was performed to find suitable dose levels of the test item and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in the toxicity of the test item between the sexes; therefore, the main test was performed using only male mice using the single administration treatment schedule for test items. The micronucleus test was conducted using the oral route in groups of seven mice (males) at the maximum recommended dose of 2000 mg/kg, with 1000 and 500 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours after dosing, the bone marrows were extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei and PCE/NCE ratio was calculated as an indicator for toxicity. Two additional groups of five mice were given a single oral dose of arachis oil, or dosed orally with cyclophosphamide, to serve as vehicle and positive controls respectively. Vehicle and positive control animals were euthanised 24 hours after dosing.

 

RESULTS

There were no premature deaths or clinical signs observed in any of the dose groups in the main test. There were no marked decreases in the PCE/NCE ratio observed in the 24 or 48-hour test item dose groups when compared to the vehicle control group. There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

 

CONCLUSION

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Ames test

The key test 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 harmonised guideline - Bacterial Reverse Mutation Test.

Salmonella typhimuriumstrains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was not amended following the results of Experiment 1 and was 1.5 to 5000 μg/plate. Eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item.

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

 

The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. The test item induced a visible reduction in the growth of the bacterial background lawns of TA100, in the absence of S9-mix, from 1500 μg/plate and at 5000 μg/plate to TA1535 (absence of S9-mix) and TA100 and TA1535 in the presence of S9-mix. No toxicity was noted to any of the remaining bacterial tester strains in either the absence or presence of S9-mix. Consequently, the same maximum dose level was used in the second mutation test. A similar toxic response was noted in Experiment 2 with weakened bacterial background lawns noted to TA100 and TA1535 at 5000 μg/plate in both the presence and absence of S9-mix. Once again, no toxicity was noted to TA98, TA1537 and WP2uvrA at any test item dose level in either the absence or presence of S9-mix. 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 substance was considered to be non-mutagenic under the conditions of the test.

Chromosome aberration test

The key study was designed to be compatible with OECD Guidelines for Testing of Chemicals No. 473 "In VitroMammalian Chromosome Aberration Test" adopted 29th July 2016 and the Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011.

 

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after 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 used in the Main Experiment were selected using data from the Cell Growth Inhibition Test (Preliminary Toxicity Test) where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the Main Test were 10, 20, 40, 80, 100, 120, 160 μg/mL (4(20)-hour without S9 exposure group), 20, 40, 80, 160, 200, 240, 320 μg/mL (4(20)-hour with S9 (2%) exposure group) and 20, 40, 80, 160, 200, 240, 320 μg/mL (24-hour without S9 exposure group).

 

All vehicle (dimethyl sulphoxide) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item demonstrated marked toxicity in all three exposure groups and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that induced approximately 50% mitotic inhibition or greater. The test item was 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 29 July 2016, 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.

 

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 8 dose levels in duplicate, together with vehicle (DMSO), 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 test item exhibited marked toxicity in all three exposure groups. Precipitate of the test item was also observed in all three of the exposure groups. The dose levels plated for viability and expression of mutant colonies were 10, 20, 40, 60, 80, 100 μg/mL (4-hour without S9 group), 40, 80, 100, 120, 140, 160 μg/mL (4-hour with S9 (2%) group) and 40, 80, 100, 120, 160, 200 μg/mL (24-hour without S9 group).

 

The maximum dose level used was limited by test item induced toxicity. No precipitate of the test item was observed throughout the main test. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. Very near to optimum levels of toxicity were considered to have been achieved in all three exposure groups. 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 Global Evaluation Factor (GEF) of 126 x 10E-06 and was considered to be non-mutagenic in this assay.

In vivo

Mouse micronucleus test

The key study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 474 “Mammalian Erythrocyte Micronucleus Test” (adopted 29 July 2016), Method B.12 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA (TSCA) OPPTS 870.5395, EPA 712-C-98-226, August 1998 guidelines, and be acceptable to the Japanese METI/MHLW/MAFF guidelines for testing of new chemical substances.

A range-finding test was performed to find suitable dose levels of the test item and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in the toxicity of the test item between the sexes; therefore, the main test was performed using only male mice using the single administration treatment schedule for test items. The micronucleus test was conducted using the oral route in groups of seven mice (males) at the maximum recommended dose of 2000 mg/kg, with 1000 and 500 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours after dosing, the bone marrows were extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei and PCE/NCE ratio was calculated as an indicator for toxicity.

Two additional groups of five mice were given a single oral dose of arachis oil, or dosed orally with cyclophosphamide, to serve as vehicle and positive controls respectively. Vehicle and positive control animals were euthanised 24 hours after dosing.

 

There were no premature deaths or clinical signs observed in any of the dose groups in the main test. There were no marked decreases in the PCE/NCE ratio observed in the 24 or 48-hour test item dose groups when compared to the vehicle control group. There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

 

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

 

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

Justification for classification or non-classification

In vitro tests demonstrated that the test material was non-mutagenic (Ames test and mouse lymphoma assay) and non-clastogenic (chromosome aberration test). An in vivo study (mouse micronucleus test) also concluded that the test item was non-genotoxic. Classification in accordance with Regulation (EC) No 1272/2008 is therefore not required.