2-Propenoic acid, 2-hydroxyethyl ester, reaction products with O,O-bis(dialkyalkyl) hydrogen phosphorodithioate and dimethyl phosphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: The test item was considered to be non-mutagenic (OECD 471, EU Method B13/B14, ICH S2(R1) and relevant Japanese guidelines)

 

Chromosome aberration test: The test item was 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 increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF), consequently it is considered to be non-mutagenic in this assay (OECD 490, EU Method B.17 and OPPTS 870.5300).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 June 2019 to 11 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

31 March 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register.

Version / remarks:

77:33748-33749

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, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Moltox post mitochondrial supernatant (S9)

Test concentrations with justification for top dose:

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

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

acetone (Acros Organics; batch 1882926; purity ≥ 99.75 %; expiry date July 2020)

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

2 μg/plate for WP2uvrA, 3 μg/plate for TA100, 5 μg/plate for TA1535 (all without S9)

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 μg/plate for TA1537 without S9

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 μg/plate for TA98 without S9

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

1 μg/plate for TA100, 2 μg/plate for TA1535 and TA1537, 10 μg/plate for WP2uvrA (all with S9)

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

5 μg/plate for TA98 with S9

Details on test system and experimental conditions:

STUDY CONTROLS
- The vehicle control used was acetone. 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 prior to Experiment 1).

MICROSOMAL ENZYME FRACTION
- The S9 Microsomal fractions (Sprague-Dawley) were purchased from Moltox.
- Lot No. PB/βNF S9 4061 was used in this study.
- A copy of the S9 Certificate of Efficacy is presented in Appendix 3 (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.

MEDIA
- Top agar was prepared using 0.6 % Bacto agar (lot numbers 7193746 04/2022 and 8255817 07/2023) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar.
- Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 51159 07/2019 and 51213 08/2019).

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 LT2 through mutations in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an inactivation of the excision repair system and a dependence on exogenous biotin. In the strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA repair deficiency which enhances its sensitivity to some mutagenic compounds. This deficiency allows the strain to show enhanced mutability as the uvrA repair system would normally act to remove and repair the damaged section of the DNA molecule (Green and Muriel, 1976 and Mortelmans and Riccio, 2000).
- The bacteria used in the test were obtained from British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987 or Trinova Biochem GmbH on 27 June 2017.
- 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 2321478 04/2023) and incubated at 37 ± 3 °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
- The test item was immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but was fully miscible in acetone at 100 mg/mL in solubility checks performed in-house. Acetone was therefore selected as the vehicle.
- The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in pre-dried acetone by mixing on a vortex mixer and sonication for 5 minutes at 40 °C. No correction for purity was required.
- All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

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, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. 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 between 48 and 72 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).

DOSE SELECTION FOR EXPERIMENT 2
- Following Sponsor recommendation the plate incorporation method was employed for Experiment 2 in the presence and absence of metabolic activation (S9-mix).
- The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 μg/plate.
- Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxicity 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 between 48 and 72 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).
- Sporadic manual counts were performed due to spreading colonies and light background contamination which prevented an accurate automated count.

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), Mortelmans and Zeiger (2000), Green and Muriel (1976) and Mortelmans and Riccio (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 2017 and 2018 are presented in Appendix 2 (attached).
(iii) All tester strain cultures should be in the range of 0.9 to 9 x 10E+09 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 2017 and 2018 are presented in Appendix 2 (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
- Perceptive Instruments – Ames Study Manager v1.24 and Ames Sorcerer v3.

Rationale for test conditions:

- The purpose of the study was to evaluate the test item for the ability to induce reverse mutations, either directly or after metabolic activation, at the histidine or tryptophan locus in the genome of five strains of bacteria.
- The study was based on the in vitro technique described by Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. The Salmonella typhimurium strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base-pair substitution to tryptophan independence (Green and Muriel, 1976 and Mortelmans and Riccio, 2000) is used to complement the Salmonella strains.
- Since many compounds do not exert a mutagenic effect until they have been metabolized by enzyme systems not available in the bacterial cell, the test item and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix) prepared from rats pre-treated with a mixture known to induce an elevated level of these enzymes.

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) 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).
(v) Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

- Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS
- 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 vehicle (acetone) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
- 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 (S9-mix), are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2. The results are also expressed graphically in Figure 1 to Figure 4. All relevant tables and figures are attached.
- A history profile of vehicle, untreated and positive control values (reference items) is presented in Appendix 2 (attached).

EXPERIMENT 1
- The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate.
- There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
- A test item precipitate (globular in appearance) was noted at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.
- There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

EXPERIMENT 2
- The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 μg/plate).
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
A test item precipitate (globular in appearance) was noted at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Conclusions:

The test item was considered to be non-mutagenic under the conditions of this 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, the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749) 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 metabolising system (10 % liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxicity of the test item.

 

RESULTS

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

 

The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).

 

Based on the results of Experiment 1, the same maximum dose level (5000 μg/plate) was employed in the second mutation test (plate incorporation method). Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).

 

A test item precipitate (globular in appearance) was noted at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. This observation did not prevent the scoring of revertant colonies.

 

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).

 

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 (plate incorporation method).

 

CONCLUSION

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

26 June 2019 to 06 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

29 July 2016

Deviations:

yes

Remarks:

source of S9 changed with no impact on results or integrity of the study (see below)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines (21 December 2015)

Deviations:

yes

Remarks:

source of S9 changed with no impact on results or integrity of the study (see below)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented 'in-house' with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % 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:

S9 microsomal fraction (see Appendices 5 and 6, attached)

Test concentrations with justification for top dose:

PRELIMINARY TEST
0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, and 320 μg/mL.

MAIN TEST
4(20) hour without S9: 0, 10, 20, 40, 60, 80 and 160 µg/mL
4 (20) hour with S9 (2 %): 0, 10, 20, 40, 60, 80 and 160 µg/mL
24 hour without S9: 0, 10, 20, 40, 60, 80 and 160 µg/mL

Vehicle / solvent:

Acetone

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Remarks:

Acros Organics; batch 1882926; purity 99.97 %; expiry date 20 November 2020

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

mitomycin C

Remarks:

Sigma Aldrich; CAS 50-07-7; batch SLBN5647V; purity of 100 % assumed; expiry date 01 October 2019; solvent Minimal Essential Medium

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

Acros Organics; CAS 6055-19-2; batch A0389646; purity 99.9 %; expiry date 01 October 2022; solvent DMSO

Details on test system and experimental conditions:

PURPOSE OF THE STUDY
- The purpose of the study was to assess the potential chromosomal mutagenicity of the test item on the metaphase chromosomes of normal human lymphocytes. Human peripheral blood lymphocytes are recognized in the OECD 473 guidelines as being a suitable cell line for the Mammalian Chromosome Aberration Test.
- Numerical and structural chromosome aberrations are implicated in the pathology of neoplasia (Radman et al. 1982; Cairns, 1981) and also occur in a high proportion of spontaneous abortions and abnormal live births (Chandley, 1981). Furthermore, most carcinogens are capable of inducing such changes in chromosome fidelity. Metaphase analysis in vitro involves recording such structural and numerical aberrations in the chromosomes of exposed cells. Many of these changes are lethal to the cells in which they occur and are therefore not of heritable significance. However, it is assumed that agents capable of inducing gross chromosomal changes also induce more subtle changes (translocations, inversions and small deletions) which are not cell lethal, and therefore represent a hazard. The ability to induce chromosome aberrations also correlates well with the induction of gene mutations (Hollstein et al. 1979).

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 female (aged 32 years) and the donor for the main experiment was female (aged 35 years).

MICROSOMAL ENZYME FRACTION AND S9-MIX
- Microsomal enzyme fraction Lot Number 28/10/18 was used in the preliminary toxicity test, and was pre-prepared in house (outside the confines of the study) following standard procedures.
- The S9 Microsomal fraction in the main test was purchased from Moltox (lot number 4061; expiry date February 2021).
- Copies of the S9 Certificates of Efficacy and Quality Control & Production Certificate, demonstrating the capability to activate known mutagens, are presented in Appendix 5 for the preliminary test (attached) and Appendix 6 for the main test (attached).
- The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20 % (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10 % volume of S9-mix into culture media, was 2 %.

TEST ITEM PREPARATION AND ANALYSIS
- The test item was considered to be a UVCB and, therefore, the maximum recommended dose was initially set at 5000 μg/mL.
- The test item was considered to have a purity of 100% and no adjustment was required in the formulations.
- The test item was insoluble in culture medium at 50 mg/mL and dimethyl sulphoxide at 500 mg/mL but was soluble in acetone at 500 mg/mL in solubility checks performed in-house.
- Due to the sensitivity of human lymphocytes to acetone, the formulations were dosed at 0.5% in 50 μL aliquots. Consequently, the maximum practical concentration was 2500 μg/mL.
- Prior to each experiment, the test item was accurately weighed, formulated in acetone 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 presented in the table below.
- A precipitate of the test item was observed at and above 78.13 μg/mL in the solubility test and therefore the maximum dose level for the Preliminary Toxicity Test was limited to 320 μg/mL.
- 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 was reflected in the GLP compliance statement.

CULTURE CONDITIONS
- Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing components.
- Quadruplicate cultures were established for the vehicle control.
- When dispensed into sterile plastic flasks each culture contained 9.05 mL MEM, 10 % (FBS) 0.1 mL Li-heparin, 0.1 mL phytohaemagglutinin and 0.75 mL heparinised whole blood

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 0.05 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. 1 mL of 20 % S9-mix (i.e. 2 % final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test (Cell Growth Inhibition Test) and Main Experiment.
- After 4 hours at approximately 37 °C, 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 ºC 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 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
- After 4 hours at approximately 37 °C, 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 0.05 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.
- The Cell growth Inhibition Test (Preliminary Toxicity Test) was performed using all three of the exposure conditions as described for the Main Experiment but using single cultures only for the test item dose levels and duplicate cultures for the vehicle control.

CELL GROWTH INHIBITION TEST (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 0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, 320 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 EXPERIMENT
- Three exposure groups were used for the Main Experiment:
(i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest.
(ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest.
(iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest.
- The dose range of test item used for all three exposure groups was 0, 10, 20, 40, 60, 80 and 160 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.

CELL HARVEST
- Mitosis was arrested by addition of demecolcine (Colcemid 0.1 μg/mL) two hours before the required harvest time.
- After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075 M 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
- Where possible 1000 cells per culture were evaluated for the incidence of metaphase cells 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, 600 from the vehicle control (150 per replicate), 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) (see 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 (including endoreduplicated cells) and the incidence of polyploid cells (%) reported. Endoreduplicated cells were recorded separately and are included in the polyploid cell total number. 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
- Delta Building Monitoring System.
- R Environment for Statistical Computing.

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.
(ii) All the positive control chemicals induced a positive response (p ≤ 0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix.
(iii) The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
(iv) The required number of cells and concentrations were analysed.

Statistics:

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

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
- The dose range for the Cell Growth Inhibition Test was 0, 1.25, 2.5, 5, 10, 20, 40, 80, 160, and 320 μg/mL. The maximum dose was limited to 320 μg/mL based on the precipitate observations made in the solubility test.
- A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 80 μg/mL, in all three exposure groups.
- Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 160 μg/mL in the 4(20)-hour exposure group in the absence of S9. The maximum dose with metaphases present in the 4(20)-hour exposure group in the presence of S9 and in the 24-hour continuous exposure was 320 μg/mL.
- The test item induced no evidence of toxicity in the 4(20)-hour exposure in the presence of S9 or in the 24-hour exposure group. The 4(20)-hour exposure group demonstrated 52% and 39% mitotic inhibition at 40 μg/mL and 60 μg/mL, respectively but this toxicity coincided with the lowest precipitating dose level.
- The selection of the maximum dose level for the main experiment was based on the lowest precipitating dose level and 160 μg/mL was selected for all three exposure groups. This allowed for any shift in precipitate between experiments.

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 precipitate was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 160 μg/mL in all three exposure groups.
- Precipitate observations were made at the end of exposure in blood-free cultures and was noted at and above 80 μg/mL levels in all three exposure groups.
- The results of the mitotic indices (MI) from the cultures after their respective exposures are presented as cell growth indices in Forms 1, 2, and 3 (attached). They confirm the qualitative observations in that no dose-related inhibition of mitotic index was observed in the 4(20)-hour exposure group in the presence of S9 or in the 24-hour exposure group. In the absence of S9, a dose-related inhibition of mitotic index was observed at 60 μg/mL and 80 μg/mL with 39 % and 47 % mitotic inhibition, respectively and the toxicity coincided with the lowest precipitating dose level as was seen in the Preliminary Toxicity Test.
- The maximum dose level selected for metaphase analysis for all three exposure groups was the lowest precipitating dose level (80 μg/mL). The 4(20)-hour exposure group in the absence of metabolic activation also achieved near optimum toxicity at 80 μg/mL.
- The chromosome aberration data are given in Forms 1, 2 and 3 (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 Figures 2, 3 and 4 of Appendix 3 (attached) and in 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 exposure groups. There was no indication of endoreduplication noted.

DOSE LEVELS OF CONTROLS AND TEST ITEM

Group	Final concentration of test item (µg/mL)
4(20)-hour without S9	0*, 10, 20, 40*, 60*, 80*, 160, MMC 0.2*
4(20)-hour with S9 (2 %)	0*, 10, 20, 40*, 60*, 80*, 160, CP 1*
24-hour without S9	0*, 10, 20, 40*, 60*, 80*, 160, MMC 0.1*

* = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

Conclusions:

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

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 plus Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 21 December 2015.

 

METHODS

Duplicate cultures of human lymphocytes, treated with the test item (quadruplicate cultures for the vehicle) were evaluated for chromosome aberrations at three 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 metabolising 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 preliminary toxicity test where the results indicated that the maximum concentration should be limited on precipitate. The dose levels selected for the Main Test were 0, 10, 20, 40, 60, 80 and 160 μg/mL (4(20)-hour without S9, 4(20)-hour with S9 (2 %) and 24-hour without S9).

RESULTS

All vehicle (Acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level. The 4(20)-hour exposure group in the absence of metabolic activation (S9) achieved near optimum toxicity at the lowest precipitating dose level with 47 % mitotic inhibition. The 4(20)-hour exposure group in the presence of S9 and the 24-hour exposure group both demonstrated very modest toxicity at the lowest precipitating dose level.

 

CONCLUSION

The test item was 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:

03 September 2019 to 24 September 2019

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)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

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

Details on mammalian cell type (if applicable):

TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus)

Metabolic activation:

with and without

Metabolic activation system:

S9 microsomal fraction supplied by Moltox (see Annex 2, attached)

Test concentrations with justification for top dose:

PRELIMINARY STUDY
- Nominal test item concentrations: 0, 1.25, 2.5, 5, 10, 20, 40, 80, 160 and 320 μg/mL

MAIN STUDY
- Nominal test item concentrations: 0, 5, 10, 20, 40, 60, 80 μg/mL (4-hour without S9)
- Nominal test item concentrations: 0, 5, 10, 20, 40, 60, 80 μg/mL (4-hour with 2 % S9)
- Nominal test item concentrations: 0, 2.5, 5, 10, 20, 40, 60 μg/mL (24-hour without S9)

Vehicle / solvent:

Acetone

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Remarks:

Sigma Aldrich (batch BCBW8635; purity treated as 100 %; expiry date 28 March 2024; solvent DMSO)

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

Acros Organics (batch A0389646; purity 99.9 %; expiry date 01 October 2022; 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
- The S9 Microsomal fraction used during the course of the study was purchased from Moltox, Lot No. 4061, Expiry February 2021. A copy of the Quality Control & Production Certificate, demonstrating the capability to activate known mutagens, is presented in Annex 2 (attached).
- The S9 mix was prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20 % S9 concentration. The final concentration of S9 when dosed at a 10 % volume of S9-mix was 2 % for the Preliminary Toxicity Test and the Mutagenicity Test.

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

TEST ITEM PREPARATION
- The test item was a UVCB, therefore the maximum proposed dose level in the solubility test was initially set at 5000 μg/mL, the maximum recommended dose level, and no correction for the purity of the test item was applied.
- Scott et al. 1991). Following solubility checks performed in-house for the Chromosome Aberration Test performed on the same test item (Covance Study No. HP09FR), the test item was found to be immiscible in culture medium at 50 mg/mL, and dimethyl sulfoxide at 500 mg/mL. The test item was fully miscible in acetone at 500 mg/mL and was considered suitable for dosing in the solubility checks. Acetone is toxic to cells at dose volumes greater than 0.5% of the total culture volume. Therefore, the test item was formulated at 500 mg/mL and dosed at 0.5 % to give the maximum achievable dose level of 2500 μg/mL. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm. The pH and osmolality readings from the Human Lymphocyte Chromosome Aberration Test are shown in the table below.
- No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This was an exception with regard to GLP and was been reflected in the GLP compliance statement.

PRELIMINARY TOXICITY TEST
- A preliminary toxicity test was performed on cell cultures at 5 x 10E+05 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10E+05 cells/mL using a 24-hour exposure period without S9. Due to the high levels of precipitate observed in the solubility test, the dose range used in the preliminary toxicity test was 1.25 to 320 μg/mL for all three of the exposure groups. Following the exposure periods, the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 10E+05 cells/mL unless the mean cell count was less than 3 x 10E+5 cells/mL in which case all the cells were maintained.
The cultures were incubated at 37 °C with 5 % CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10E+05 cells/mL in R20 unless the mean cell count was less than 3 x 10E+05 cells/mL in which case all the cells were maintained,. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent solvent control performed to give a percentage Relative Suspension Growth (%RSG) value.
- Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
(i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 μL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
(ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
(iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

MUTAGENICITY TEST
- Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10E+06 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 10E+06 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation.
- The exposures were performed in duplicate (A + B), both with and without metabolic activation (2 % S9 final concentration) at six dose levels of the test item (1.25 to 80 μg/mL in all three of the exposure groups), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.1 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).
- The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

MEASUREMENT OF SURVIVAL, VIABILITY AND MUTANT FREQUENCY
- At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10E+05 cells/mL unless the mean cell count was less than 3 x 10E+05 cells/mL in which case all the cells were maintained. The cultures were incubated at 37 °C with 5 % CO2 in air and sub-cultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/mL unless the mean cell count was less than 3 x 10E+05 cells/mL in which case all the cells were maintained.
- On Day 2 of the experiment, the cells were counted, diluted to 10E-06
cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well 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 solvent control, and when combined with the Cloning Efficiency (%V) data, a Relative Total Growth (RTG) value.

PLATE SCORING
- Plates (96 well) 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 mutant 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 mutant plates. The plates were incubated for two hours. MTT is a vital stain that is taken up by viable cells and metabolized 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) * (48-hour cell count/2)
(ii) 24-Hour Suspension Growth (SG) = (0-hour cell count/1.5) * (24-hour cell count/2) x (48-hour cell count/2)
(iii) Day 0 Factor = dose 0-hour cell count/vehicle control 0-hour cell count
(iv) %RSG = [(dose SG x dose Day 0 Factor)/vehicle control SG] x 100

CALCULATION OF DAY 2 VIABILITY (%V)
- Since the distribution of colony-forming units over the wells is described by the Poisson distribution, the day 2 viability (%V) was calculated using the zero term of the Poisson distribution [P(0)] method:
(i) P(0) = number of negative wells / total wells plated
(ii) %V = -ln P(0) x 100 / number of cells/well

CALCULATION OF RELATIVE TOTAL GROWTH (RTG)
- For each culture, the relative cloning efficiency, RCE, was calculated using the equation RCE = %V / mean solvent control %V
- The RTG was then calculated for each culture using the equation RTG = (RCE * RSG) / 100

CALCULATION OF MUTATION FREQUENCY (MF)
- The mutation frequency (MF) per survivor = [(-ln P(0) selective medium)/cells per well in selective medium)]/surviving fraction in non-selective medium.
- The experimental data was analysed using a dedicated computer program, Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989). The statistical package used indicates the presence of statistically significant increases and linear-trend events.

DATA EVALUATION
- The current Historical Vehicle and Positive Control Mutation Frequencies are presented in Appendix 2 (attached). The Historical Vehicle and Positive Control data is generated by the Mutant 240C program on a rolling system of the last twenty sets of archived data. The program combines the 4-hour and 24-hour data in the absence of metabolic activation as the acceptability criteria is the same for all three of the exposure groups.
- Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20 % survival (80 % toxicity), but no less than 10 % survival (90 % toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
- An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10E-06, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

ACCEPTABILITY OF ASSAY
- A mutation assay is considered acceptable if it meets the following criteria (the current recommendations of the IWGT are considered; (Moore et al., 2002; Moore et al., 2003; Moore et al., 2006; Moore et al., 2007):
(1) For non-toxic test items the upper test item concentrations will be 10mM, 2 mg/mL or 2μL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCBs) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL. Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point. In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) and/or percentage Relative Suspension Growth (%RSG) to approximately 10 to 20 % of survival.
(2) The absolute Cloning Efficiency (%V) at the time of mutant selection of the solvent controls is 65 to 120 %.
(3) The total suspension growth of the solvent control following 4 hour exposure, calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold increase in cell number, should be in the range of 8 to 32. Following 24-hour exposure the total suspension growth should be in the range 32 to 180.
(4) The in-house historical solvent control mutant frequency is in the range of approximately 50 to 170 x 10E-06 cells. Solvent control results should ideally be within this range, although minor errors in cell counting and dilution, or exposure to the metabolic activation system, may cause this to be slightly elevated.
(5) Every test should also be evaluated as to whether the positive controls (EMS and CP) meet at least one of the following two acceptance criteria developed by the IWGT workgroup:
(i) The positive control should demonstrate an absolute increase in total MF, that is, an increase above the spontaneous background MF [an induced MF (IMF)] of at least 300 x 10E-06. At least 40 % of the IMF should be reflected in the small colony MF.
(ii) The positive control has an increase in the small colony MF of at least 150 x 10E-06 above that seen in the concurrent untreated/solvent control (a small colony IMF of 150 x 10E-06).
(6) The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures i.e. the Relative Total Growth (RTG) and percentage Relative Suspension Growth (%RSG) should be greater than approximately 10 % of the concurrent selective control group.
(7) A minimum of four analysed duplicate dose levels is considered necessary in order to accept a single assay for evaluation of the test item.

MAJOR COMPUTERISED SYSTEMS
- York Electronics Mutant program version 2.40 was used to process and statistically analyse the cell count and plate count data.

Evaluation criteria:

See above

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True 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 1.25 to 320 μg/mL. The results for the Relative Suspension Growth (%RSG) were as shown in the attached table.
- There was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in all three of the exposure groups. The most marked reductions in the 4-hour exposure groups in both the absence and presence of metabolic activation were only observed at dose levels beyond the onset of precipitate observed at 80 μg/mL. However, in the 24-hour exposure group in the absence of metabolic activation the most marked reductions were observed at dose levels at and beyond the onset of precipitate observed at 80 μg/mL. Therefore, the maximum dose levels in the Mutagenicity Test were limited by the presence of precipitate in the 4-hour exposure groups, in both the absence and presence of metabolic activation, and limited by a combination of precipitate and test item-induced toxicity in the 24-hour exposure group in the absence of metabolic activation, as recommended by the OECD 490 guideline
 
MUTAGENICITY TEST
- A summary of the results from the test is presented in Table 1 (attached).
- The results of the 96-well plate counts and their analysis are presented in Tables 2 to 10 (attached).
- There was evidence of marked dose related toxicity following exposure to the test item in the 24-hour exposure group in the absence of metabolic, and very modest toxicity in the 4-hour exposure group in the absence of metabolic activation, as indicated by the %RSG and RTG values (see Tables 3 and 9, attached).
- Precipitate of the test item was observed at the maximum dose level of 80 μg/mL in the 4-hour exposure groups in both the absence and presence of metabolic activation, and at 60 and 80 μg/mL in the 24-hour exposure group in the absence of metabolic activation. Therefore, following the recommendation of the OECD guideline, the lowest precipitating dose level of 60 μg/mL in the 24-hour exposure group in the absence of metabolic activation was plated for cloning efficiency and 5-TFT resistance and the subsequent maximum recommended dose level was discarded as it was considered surplus to requirements. Based on the RTG and / or %RSG values observed, optimum levels of toxicity were considered to have been achieved in the 24-hour exposure group in the absence of metabolic activation as recommended by the OECD guideline (see Table 9, attached). Acceptable levels of toxicity were seen with the positive control substances (see Tables 3, 6, and 9, attached).
- The solvent 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 at any of the dose levels in the main test, using a dose range that included the lowest precipitating dose level in all three exposure groups (optimum levels of toxicity were also achieved at the lowest precipitating dose level in the 24-hour exposure group), as recommended by the OECD 490 guideline.
- 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 theGlobal Evaluation Factor (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 Guideline for Testing of Chemicals No 490 "In Vitro Mammalian 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, and the US EPA OPPTS 870.5300 Guideline.

 

METHODS

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels in duplicate, together with vehicle (acetone) and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2 % S9), and a 24 hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were 5, 10, 20, 40, 60 and 80 μg/mL for 4-hour without S9 and 4-hour with S9 (2 %). The dose levels plated for viability and expression of mutant colonies were 2.5, 5, 10, 20, 40 and 60 μg/mL for 24-hour without S9.

 

RESULTS

The maximum dose levels in the Mutagenicity Test were limited by the presence of precipitate in the 4-hour exposure groups, in both the absence and presence of metabolic activation, and limited by a combination of precipitate and test item-induced toxicity in the 24-hour exposure group in the absence of metabolic activation, as recommended by the OECD 490 guideline. The solvent control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, using a dose range that included the lowest precipitating dose level in all three exposure groups (optimum levels of toxicity were also achieved at the lowest precipitating dose level in the 24-hour exposure group), as recommended by the OECD 490 guideline.

 

CONCLUSION

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Negative results were obtained during investigation of in vitro gene mutation in bacteria (Ames test), in vitro cytogenicity in mammalian cells (chromosome aberration study) and in vitro gene mutation in mammalian cells (mouse lymphoma assay). As a result, and in accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 6.0; July 2017), the substance is not considered to be genotoxic and no further testing is required.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro

Ames test

The key study 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, the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749) and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

 

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using the Ames plate incorporation method at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10 % liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 μg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxicity of the test item.

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

 

The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).

Based on the results of Experiment 1, the same maximum dose level (5000 μg/plate) was employed in the second mutation test (plate incorporation method). Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).

 

A test item precipitate (globular in appearance) was noted at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix) in Experiments 1 and 2. This observation did not prevent the scoring of revertant colonies.

 

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method).

 

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 (plate incorporation method).

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

Chromosome aberration test

In a key study, 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 plus Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 21 December 2015.

 

Duplicate cultures of human lymphocytes, treated with the test item (quadruplicate cultures for the vehicle) were evaluated for chromosome aberrations at three 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 metabolising 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 preliminary toxicity test where the results indicated that the maximum concentration should be limited on precipitate. The dose levels selected for the Main Test were 0, 10, 20, 40, 60, 80 and 160 μg/mL (4(20)-hour without S9, 4(20)-hour with S9 (2 %) and 24-hour without S9).

All vehicle (Acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level. The 4(20)-hour exposure group in the absence of metabolic activation (S9) achieved near optimum toxicity at the lowest precipitating dose level with 47 % mitotic inhibition. The 4(20)-hour exposure group in the presence of S9 and the 24-hour exposure group both demonstrated very modest toxicity at the lowest precipitating dose level.

 

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 Guideline for Testing of Chemicals No 490 "In Vitro Mammalian 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, and the US EPA OPPTS 870.5300 Guideline.

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at six dose levels in duplicate, together with vehicle (acetone) and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2 % S9), and a 24 hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were 5, 10, 20, 40, 60 and 80 μg/mL for 4-hour without S9 and 4-hour with S9 (2 %). The dose levels plated for viability and expression of mutant colonies were 2.5, 5, 10, 20, 40 and 60 μg/mL for 24-hour without S9.

 

The maximum dose levels in the Mutagenicity Test were limited by the presence of precipitate in the 4-hour exposure groups, in both the absence and presence of metabolic activation, and limited by a combination of precipitate and test item-induced toxicity in the 24-hour exposure group in the absence of metabolic activation, as recommended by the OECD 490 guideline. The solvent control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolising system. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, using a dose range that included the lowest precipitating dose level in all three exposure groups (optimum levels of toxicity were also achieved at the lowest precipitating dose level in the 24-hour exposure group), as recommended by the OECD 490 guideline.

 

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), consequently it is considered to be non-mutagenic in this assay.

Justification for classification or non-classification

Negative results were obtained during investigation of in vitro gene mutation in bacteria (Ames test), in vitro cytogenicity in mammalian cells (chromosome aberration study) and in vitro gene mutation in mammalian cells (mouse lymphoma assay). As a result, and in accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 6.0; July 2017), the substance is not considered to be genotoxic, no further testing is required, and classification under the terms of Regulation (EC)No. 1272/2008 does not apply.