bis(branched-alkyl) 2-{[(1-phenylethyl)amino]methyl}alkanedioate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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

In the OECD Guideline 473 In Vitro Mammalian Chromosome Aberration Test, the test item did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the presence or absence of a liver enzyme metabolising system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.

In the OECD 490 Mouse Lymphoma Assay, the test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 January 2015 to 20February 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline. There were no deviations (unplanned changes) from the study plan.

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

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:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

OPPTS harmonised guidelines

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Not required

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Non-mammalian study

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Non-mammalian study

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

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

Experiment 2: Main test: 15, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The substance was not misicible in water and but was miscible in DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent - DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

2, 3, 5 µg/plate respectively for WP2uvrA, TA100, TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent - DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

80 µg/plate for TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent - DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

0.2 µg/plate for TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent - DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

1, 2, 10 µg/plate for TA100, TA1535&TA1537, WP2uvrA respectively

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent - DMSO

True negative controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

5 µg/plate for TA98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) at multiple dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors).

RANGE FINDING
Dose selection
The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent or appropriate positive control was added to 2 mL of molten trace amino-acid supplemented media containing 0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described previously (see 3.5.1.2) except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten trace amino-acid supplemented media instead of phosphate buffer.
Incubation and Scoring
All of the plates were incubated at 37 °Ci 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).

MAIN TEST
Dose selection
The dose range used for the main test was determined by the results of the range-finding test and was 15 to 5000 µg/plate.
Six test item dose levels were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the results from the first mutation test.
Without Metabolic Activation
The procedure was the same as described previously
With Metabolic Activation
The procedure was the same as described previously
Incubation and Scoring
All of the plates were incubated at 37 °C +/- 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

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


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


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

NUMBER OF CELLS EVALUATED:
All strains 0.9 to 9 * 10>9

DETERMINATION OF CYTOTOXICITY
- Method: N/A

OTHER EXAMINATIONS:
N/A


OTHER:
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.
In order to select appropriate dose levels for use in the main test, a preliminary assay was carried out to determine the toxicity of the test material.

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

Evaluation criteria:

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

Statistics:

MAHON, G.A.T., et al (1989). Analysis of data from microbial colony assays. In: KIRKLAND D.J., (eds.). Statistical Evaluation of Mutagenicity Test Data: UKEMS sub-committee on guidelines for mutagenicity testing. Cambridge University Press Report, pp. 26-65.

Key result

Species / strain:

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

valid

Key result

Species / strain:

E. coli WP2 uvr A

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The maximum dose level of the test item in the first mutation test was selected as the maximum recommended dose level of 5000 µg/plate. In both the range-finding and main tests there was no visible reduction in the growth of the bacterial background lawns noted at any dose level, either in the presence or absence of S9-mix. A testitem precipitate (globular in appearance) was noted at 5000 µ

g/plate, this observation did not prevent the scoring of revertant colonies.

Results from the second mutation test showed the test item inducing toxicity as a weakening of the bacterial background lawns of all of the tester strains dosed in the absence of S9 at the upper test item dose levels. In the presence of S9-mix, weakened lawns were noted to TAl535 and TA100 at 5000 µg/plate. A test item precipitate (greasy/globular in appearance) was observed at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the first mutation test. 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 S9-mix in the second mutation test. A small, statistically significant increase in TA100 revertant colony frequency was observed in the presence of S9 -mix at 50 ug/plate in the main test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 50 ug/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.3 times the concurrent vehicle control.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Conclusions:

Interpretation of results (migrated information):
negative

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

Executive summary:

Introduction

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 TAl53 5, TA1537, TA98 and TAl00 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 the range-finding test was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended, following the results of the range-findi ng test, and was 15 to 5000 µg/plate.

Six test item dose levels were selected in the main test in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the results from the first mutation test.

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 and second experiments was selected as the maximum recommended dose level of 5000 µg/plate. In both the range-finding and main tests there was no visible reduction in the growth of the bacterial background lawns noted at any dose level, either in the presence or absence of S9-mix . A test item precipitate (globular in appearance) was noted at 5000 ug/plate, this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the first mutation test. 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 S9-mix in the second mutation test. A small, statistically significant increase in TA100 revertant frequency was observed in the presence of S9 -mix at 50 ug/plate in the main test. This increase was considered to be of no biologicasl relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 50 ug/plate were within in-house historical untreated/vehicle control range for the tester strain and the fold-increase was only 1.3 times the concurrent vehicle control.

Conclusion

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

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 January 2018 to 05 February 2018

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

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:

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

Test concentrations with justification for top dose:

- Preliminary cytotoxicity test: 0, 1.22, 2.44, 4.88, 9.75, 19.5, 39,. 78, 156, 312 μg/mL
- Main experiment: 5, 10, 20, 30, 40, 60 μg/mL (4-hour without S9)
- Main experiment: 10, 20, 40, 60, 80, 120 μg/mL (4-hour with 2 % S9)
- Main experiment: 10, 20, 30, 40, 60, 80 μg/mL (24-hour without S9).

Vehicle / solvent:

Acetone

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Positive controls:

yes

Remarks:

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

Positive control substance:

ethylmethanesulphonate

Remarks:

Sigma (batch BCBW8635; purity treated as 100 %; expiry 10 January 2024)

Positive controls:

yes

Remarks:

CP in DMSO 1.5 μg/mL with metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

Acros Organics (batch A0389646; purity 97 %; expiry 01 October 2022)

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 numbers PB/βNF S9 31/08/18 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).
- S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.
- The 20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and Mutagenicity Test.

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

TEST ITEM PREPARATION
- 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.
- The test item was found to be insoluble in R0 Medium at 50 mg/mL, and dimethyl sulfoxide at 500 and 250 mg/mL. The test item formed a solution in acetone at 500 mg/mL considered acceptable for dosing in the solubility checks. Acetone is toxic to L5178Y cells at dose volumes greater than 0.5% of the total culture volume. Therefore, the test item was formulated at 500 mg/mL and dosed at 0.5% to give 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 (Scott et al. 1991). - The pH and osmolality readings are presented in the table below.
- The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. 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. Due to the high levels of precipitate observed in the solubility test, the dose range used in the preliminary toxicity test was 1.22 to 312 μg/mL for all three of the exposure groups. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.
- The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.
- Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:
(i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 μL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
(ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
(iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

MUTAGENICITY TEST
- Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 106 cells/mL in 10 mL cultures were established in 25 cm2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (2.5 to 80 μg/mL in the 4-hour and 24-hour exposure groups in the absence of metabolic activation, and 5 to 160 μg/mL in the 4-hour exposure group in the presence of metabolic activation), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 0.1 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).
- The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.

MEASUREMENT OF SURVIVAL, VIABILITY AND MUTANT FREQUENCY
- At the end of the exposure periods, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10E05 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10E05 cells/mL, unless the mean cell count was less than 3 x 10E05 cells/mL in which case all the cells were maintained.
- On Day 2 of the experiment, the cells were counted, diluted to 10E04 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
- The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post exposure toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

PLATE SCORING
- Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37 °C with 5 % CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded as the additional information may contribute to an understanding of the mechanism of action of the test item (Cole et al, 1990).
- Colonies 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

DATA EVALUATION
- The current Historical Vehicle and Positive Control Mutation Frequencies are presented in Appendix 1 (attached). The Historical Vehicle and Positive Control data is generated by the Mutant 240C program on a rolling system of the last twenty sets of archived data. The program combines the 4-hour and 24-hour data in the absence of metabolic activation as the acceptability criteria is the same for all three of the exposure groups.
- Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
- An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

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

MAJOR COMPUTERISED SYSTEMS
- Delta Building Monitoring System.

Evaluation criteria:

See above

Statistics:

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

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

acetone

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 1.22 to 312 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, when compared to the concurrent vehicle control groups. Precipitate of the test item was observed at and above 78 μg/mL in the 4-hour and 24-hour exposure groups in the absence of metabolic activation, and at and above 156 μg/mL in the 4-hour exposure group in the presence of metabolic activation. It should be noted that the toxicity marginally exceeded optimum levels of toxicity at the lowest precipitating dose level in all three of the exposure groups. Therefore, the maximum dose levels in the Mutagenicity Test were limited by a combination of the onset of test item precipitate and test item-induced toxicity in all three of the exposure groups, 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 microtitre 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 all three of the exposure groups, as indicated by the %RSG and RTG values (Tables 3, 6, and 9). There was also evidence of modest reductions in viability (%V) in all three of the exposure groups, indicating that residual toxicity had occurred (Tables 3, 6, and 9). Based on the %RSG and RTG values observed, optimum levels of toxicity were achieved in all three of the exposure groups (see Tables 3, 6, and 9, attached). Optimum levels of toxicity were achieved at the precipitating dose level in the 24-hour exposure group in the absence of metabolic activation, and the dose level immediately before the onset of precipitate in the 4-hour exposure groups in both the absence and presence of metabolic activation where excessive toxicity was also observed. Therefore, the precipitating dose levels where excessive toxicity was also observed were not plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with the positive control substances (see Tables 3, 6, and 9, attached).
- At the end of the exposure periods, the onset of precipitate of the test item was observed at 80 μg/mL in the 4-hour and 24-hour exposure groups in the absence of metabolic activation, and 160 μg/mL in the 4-hour exposure group in the presence of metabolic activation.
- The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional (see Tables 3, 6, and 9, attached).
- The test item did not induce any toxicologically significant or dose related increases in the mutant frequency x 10E-06 per viable cell at any of the dose levels, in any of the three exposure groups.
- The numbers of small and large colonies and their analysis are presented in Tables 4, 7, and 10 (attached).

Conclusions:

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

Executive summary:

GUIDELINE

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline 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, 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 eight dose levels in duplicate, together with vehicle (acetone) and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were 5, 10, 20, 30,l 40 and 60 μg/mL for 4-hour without S9, 10, 20, 40, 60, 80 and 120 μg/mL for 4-hour with S9 (2%) and 10, 20, 30, 40, 60, and 80 μg/mL for 24-hour without S9.

 

RESULTS

The maximum dose levels in the Mutagenicity Test were limited by a combination of the onset of test item precipitate and test item-induced toxicity in all three of the exposure groups, as recommended by the OECD 490 guideline. The vehicle 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 metabolizing system. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

 

CONCLUSION

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

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 February 2015 to 24 April 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with recognised guideline

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:

in vitro mammalian chromosome aberration test

Target gene:

N/A

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum, at approximately 37 °C with 5% CO2 in humidified air.
- Properly maintained: NDA
- Periodically checked for Mycoplasma contamination: NDA
- Periodically checked for karyotype stability: NDA
- Periodically "cleansed" against high spontaneous background: NDA

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Preliminary Experiment
19.53 to 5000 µg/mL in three exposure groups.

Main Experiment
0, 4.88, 9.75, 19.5, 29.25, 39, 58.5 and 78 µg/mL 4-hour treatment without S9
0, 9.75, 19.5, 39, 78, 117, 156 and 312 µg/mL 4-hour treatment with S9
0, 4.88, 9.75, 19.5, 39, 58.5 and 78 µg/mL 24-hour treatment without S9

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble at 50 mg/mL in MEM but was soluble in DMSO at 500 mg/mL.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In absence of S9, 0.4 µg/mL in the 4(20)-hour exposure, 0.2 µg/mL in the 24-hour continuous exposure

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In presence of S9, 5 µg/mL

Details on test system and experimental conditions:

- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented "in-house" with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum.
- Properly maintained: NDA
- Periodically checked for Mycoplasma contamination: NDA
- Periodically checked for karyotype stability: NDA
- Periodically "cleansed" against high spontaneous background: NDA

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

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

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

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

NUMBER OF REPLICATIONS: Treatments performed in duplicate.

NUMBER OF CELLS EVALUATED: Where possible the first 150 consecutive well-spread metaphases from each culture were scored for chromosome aberrations.

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

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes in comparison to controls
- Determination of endoreplication: Yes. If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as endoreduplicated.

Evaluation criteria:

A test item can be classified as non-genotoxic if:

1. The number of induced chromosome aberrations in all evaluated groups is within the range of historical control data.
2. No toxicologically or statistically significant increase in the number of 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 induced chromosome aberrations in all evaluated groups is outside the range of historical control data.
2. At least one concentration exhibits a statistically significant increase in the frequency of cells with aberrations compared to the concurrent negative control.
3. The observed increase in the frequency of cells with aberrations is considered to be dose-related.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Key result

Species / strain:

lymphocytes:

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/mL.
The maximum dose was based on the maximum recommended dose level. A precipitate of the test item was observed at and above 156.25 µg/mL in all exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at up to 78.13 µg/mL in the 4 and 24 hours’ exposure without metabolic activation and at up to 156.25 µg/mL with metabolic activation. The selection of the maximum dose level for the Main Experiment was based on toxicity for the 4 hour exposure groups and the continuous exposure group.

Main Experiment
- A precipitate of the test item was observed at 312 µg/mL in the presence of S9-mix only.
- Inhibition of mitotic index was observed and was related to the exposure conditions; in the 4(20)-hour exposure group dosed in the absence of S9, 33%, 29%, 74% and 75% mitotic inhibition was achieved at 29.25, 39, 58.5 and 78 µg/ml respectively.
- In the 4(20)-hour exposure group dosed in the presence of S9-mix, 27%, 77% and 81% mitotic inhibition was achieved at 78, 117 and 156 µg/mL respectively.
- In the 24-hour exposure group, 29 and 57% mitotic inhibition was noted at 58,5 and 78 µg/mL respectively.

- The maximum dose level selected for metaphase analysis was the concentration closest to optimum toxicity.
- Where this was not achieved dose levels below and above the optimum toxicity limit were selected.
- The maximum concentrations selected for analysis were 58.5 µg/mL in the 4(20)-hour exposure in the absence of S9, 117 µg/mL in presence of S9 and 78 µg/mL in the 24-hour exposure.

- All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control items induced statistically significant increases in the frequency of cells with aberrations indicating that the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item did not induce any statistically significant increases in the frequency of cells with aberrations, either in the absence or presence of metabolic activation. There was no significant increase in the incidence of polyploidy at any dose level in any of the exposure groups.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

See attached background material.

Conclusions:

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

Executive summary:

Introduction

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

 

Method

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 using a 4-hour exposure in the absence and presence of a standard metabolizing system (S9 at a 2% final concentration) 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 by toxicity. The dose levels selected for the Main Test were as follows.

 

Group                                     Final concentration of test (µg/mL)

4(20)-hour without S9           0, 4.88, 9.75, 19.5, 29.25, 39, 58.5, 78

4(20)-hour with S9 (2%)        0, 9.75, 19.5, 39, 78, 117, 156, 312

24-hour without S9                0, 4.88, 9.75, 19.5, 39, 58.5, 78

 

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. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberrations using a dose range that included a dose level that approached optimum toxicity.

 

Conclusion

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

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.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial Mutagenicity Assay

Introduction

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 TAl53 5, TA1537, TA98 and TAl00 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 the range-finding test was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterialstrains and fresh test item formulations. The dose range was amended, following the results of the range-findi ng test, and was 15 to 5000 µg/plate. Six test item dose levels were selected in the main test in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the results from the first mutation test.

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 and second experiments was selected as the maximum recommended dose level of 5000 µg/plate. In both the range-finding and main tests there was no visible reduction in the growth of the bacterial background lawns noted at any dose level, either in the presence or absence of S9-mix . A test item precipitate (globular in appearance) was noted at 5000 ug/plate, this observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in the first mutation test. 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 S9-mix in the second mutation test. A small, statistically significant increase in TA100 revertant frequency was observed in the presence of S9 -mix at 50 ug/plate in the main test. This increase was considered to be of no biologicasl relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant colony counts at 50 ug/plate were within in-house historical untreated/vehicle control range for the tester strain and the fold-increase was only 1.3 times the concurrent vehicle control.

Conclusion

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

Mouse Lymphoma Assay

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline 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, 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 eight dose levels in duplicate, together with vehicle (acetone) and positive controls using 4 hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 hour exposure group in the absence of metabolic activation. The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were 5, 10, 20, 30,l 40 and 60 μg/mL for 4-hour without S9, 10, 20, 40, 60, 80 and 120 μg/mL for 4-hour with S9 (2%) and 10, 20, 30, 40, 60, and 80 μg/mL for 24-hour without S9.

 

Results

The maximum dose levels in the Mutagenicity Test were limited by a combination of the onset of test item precipitate and test item-induced toxicity in all three of the exposure groups, as recommended by the OECD 490 guideline. The vehicle 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 metabolizing system. The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Conclusion

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

Chromosome aberration test

Introduction

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

 

Method

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 using a 4-hour exposure in the absence and presence of a standard metabolizing system (S9 at a 2% final concentration) 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 by toxicity. The dose levels selected for the Main Test were as follows.

 

Group                                     Final concentration of test (µg/mL)

4(20)-hour without S9           0, 4.88, 9.75, 19.5, 29.25, 39, 58.5, 78

4(20)-hour with S9 (2%)        0, 9.75, 19.5, 39, 78, 117, 156, 312

24-hour without S9               0, 4.88, 9.75, 19.5, 39, 58.5, 78

 

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. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberrations using a dose range that included a dose level that approached optimum toxicity.

 

Conclusion

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

Justification for classification or non-classification

Harmonized classification:

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

Self classification:

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