Phenol, mono-C24-28 (even)-sec-alkyl derivs, ortho- and para-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: The experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test item demonstrated no mutagenic activity in the bacterial tester strains used in the study (OECD 471, EU Method B13/14, OPPTS 870.5100 and relevant Japanese guidelines).

 

Chromosome aberration test: The test item did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, the test item was not considered clastogenic in this test system (OECD 473 and EU Method B.10).

 

Mouse lymphoma assay: Treatment with the test item did not result in a statistically significant or biologically relevant increase in the mutation frequency in the presence and absence of a rat metabolic activation system (S9 fraction). Therefore, no mutagenic activity of the test item was concluded in the performed experiments (OECD 490 and EU Method B.17).

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 November 2016 to 25 November 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

complementary test with TA1537 necessary with no impact on integrity or results of the experiment (see below)

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

complementary test with TA1537 necessary with no impact on integrity or results of the experiment (see below)

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

complementary test with TA1537 necessary with no impact on integrity or results of the experiment (see below)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

yes

Remarks:

complementary test with TA1537 necessary with no impact on integrity or results of the experiment (see below)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

Test concentrations with justification for top dose:

- Preliminary concentration range-finding test: 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate
- Initial Mutation Test, Confirmatory Mutation Test and Complementary Initial Mutation Test in Salmonella typhimurium TA1537 strain with and without metabolic activation: 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Vehicle / solvent:

Acetone

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

other: 4-nitro-1,2-phenylenediamine

Remarks:

NPD (4 μg/plate for Salmonella TA98)

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

sodium azide

Remarks:

SAZ (2 μg/plate for Salmonella TA100 and TA1535)

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

9-aminoacridine

Remarks:

9AA (50 μg/plate for Salmonella TA1537)

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

methylmethanesulfonate

Remarks:

MMS (2 μL/plate for E.coli WP2 uvrA)

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA (2 μg/plate for all Salmonella strains)

Positive control substance:

other: 2-aminoanthracene

Remarks:

2AA (50 μg/plate for E.coli WP2 uvrA)

Negative solvent / vehicle controls:

yes

Positive control substance:

other: Acetone

Negative solvent / vehicle controls:

yes

Positive control substance:

other: Dimethyl sulfoxide

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Positive control substance:

other: Distilled water

Details on test system and experimental conditions:

FORMULATION
- The appropriate vehicle and the behaviour of the test item formulations with the solution of top agar and phosphate buffer were determined in a preliminary compatibility test.
- All dilutions in the main tests of test item were made in the testing laboratory using Acetone (approx. 12 minutes ultrasonic water bath).
- Test solutions were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock solution using the selected solvent.
- Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.
- No purity conversion was applied in the study.
- The respective test concentrations in the main tests are shown in Table 1 (below).

POSITIVE CONTROLS
- Strain specific positive controls were included in the assay, which demonstrated the effective performance of the test.
- Positive control materials were selected based on the scientific literature, the experience of the Test Facility and the availability of historical control data.
- 2-aminoanthracene (2AA) Batch number of 10157819 was used in the Preliminary Concentration Range Finding Test and batch number of STBD3302V was used in the Main tests.

VEHICLE/SOLVENT CONTROLS
- Three vehicle (solvent) control groups were used depending on the solubility of the test item and the solubility of strain specific positive chemicals.

BACTERIAL STRAINS
- Source: All bacterial strains were received from MOLTOX (Molecular Toxicology Inc, Boone, North Carolina, USA) on 21 April 2015. True copies of original certificates plus other strain documentation were collected and stored in the Microbiological Laboratory of CiToxLAB Hungary Ltd.
- Genotypes: In addition to histidine or tryptophan mutation, each strain has additional mutations,
which enhances its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair, making them more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The presence of rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules.
- The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2 uvrA) is also defective in DNA excision repair. The genotypes of the tester strains used for mutagenicity testing are summarised in Table 3 (attached).
- Storage: The strains were stored at -80 ± 10ºC in the Culture Collection of the Microbiological Laboratory of CiToxLAB Hungary Ltd. Frozen permanent cultures of the tester strains were prepared from fresh, overnight cultures to which DMSO was added as a cryoprotective agent.
- Confirmation of phenotype: The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al. and Maron and Ames. Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture, raw data and reports of phenotype confirmation are stored in the Microbiological Laboratory of CiToxLAB Hungary Ltd.

SPONTANEOUS REVERSION OF TESTER STRAINS
- Each test strain reverts spontaneously at a frequency that is characteristic of the strain. Spontaneous reversion of the test strains to histidine (Salmonella typhimurium strains) or tryptophan (in Escherichia coli strain) independence is measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.
- Historical control values for spontaneous revertants (revertants/plate) for untreated control sample without metabolic activation were in the period of 2011 to 2014 were (as guide) as follows: Salmonella typhimurium TA98: 9-46, TA100: 54-210, TA1535: 1-46, TA1537: 1-24, Escherichia coli WP2 uvrA: 11-82. More detailed historical
control data are shown in Appendix 7 (attached).

PROCEDURE FOR GROWING CULTURES
- The day before treatment, the frozen bacterial cultures were thawed at room temperature and 200 μL inoculum were used to inoculate each 50 mL of Nutrient Broth No.2 for the overnight cultures in the assay.
- The cultures were incubated for 10-14 hours at 37 °C in a Gyrotory Water Bath Shaker.

VIABILITY OF THE TESTING CULTURES
- The viability of each testing culture was determined by plating 0.1 mL of the 10E05, 10E06, 10E07 and 10E08 dilutions prepared by sterile physiological saline on nutrient agar plates.
- The viable cell number of the cultures was determined by manual counting after approximately 24-hour incubation at 37 °C.

MEDIA
- The supplier, batch number and expiry date of chemicals used in the investigation is summarised in Table 5 (attached).
- Minimal glucose agar typically contained glucose (20.0 g/L); magnesium sulfate (0.2 g/L); citric acid (2.0 g/L); dipotassium hydrogenphosphate (10.0 g/L); sodium ammonium hydrogenphosphate (3.5 g/L); agar (13.0 g/L); distilled water of a quantity sufficient to give a volume of 1000 mL.
- Minimal glucose agar plates were provided by Merck (Batch number: 140464, Expiry date: 10 January 2017 was used in the Preliminary Concentration Range Finding Test; Batch number: 141537, Expiry date: 21 March 2017 was used in the Initial Mutation Test, in the Complementary Initial Mutation Test and Confirmatory Mutation Test). Certificate of Analysis was obtained from the Supplier.
- Nutrient Broth No 2 (25.0 g made up to a volume of 1000 mL with distilled water) was sterilised in an autoclave at 121 °C.
- Nutrient agar (20.0 g made up to a volume of 1000 mL with distilled water) was sterilised in an autoclave at 121 °C.
- Top agar for Salmonella typhimurium strains contained agar bacteriological (4.0 g); NaCl (5.0 g); sufficient distilled water to give a volume of 1000 mL. Sterilisation was performed at 121 °C in an autoclave.
- Histidine-biotin solution (0.5 mM) contained D-biotin (FW 244.31) 122.2 mg; L-histidine HCl:H2O (FW 209.63) 104.8 mg; sufficient distilled water to give a volume of 1000 mL. Sterilisation was performed by filtration using a 0.22 µm membrane filter.
- Complete top agar for Salmonella typhimurium strains contained 0.5 mM histidine-biotin solution (100 mL) and agar solution (900 mL).
- Trptophan solution (2 mg/mL) contained L-tryptophan (FW 204.23) 2000 mg and sufficient distilled water to give a volume of 1000 mL. Sterilisation was performed by filtration using a 0.22 µm membrane filter.
- Complete top agar for the Escherichia coli strain contained Nutrient Broth No 2 (50 mL), 2 mg/mL tryptophan solution (2.5 mL) and agar solution 947.5 mL.

METABOLIC ACTIVATION SYSTEM
- Test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial S9 fraction.
- The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of CiToxLAB Hungary Ltd. according to Ames et al. and Maron and Ames.
- The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.
- The supplier, batch number and expiry date of chemicals used are summarised in Table 5 (attached).

INDUCTION OF LIVER ENZYMES
- Male Wistar rats (345-441 g, animals were 9 weeks old at the initiation) were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days.
- Rats were given drinking water and food ad libitum until 12 h before sacrifice when food was removed.
- Sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels.
- Initiation of the induction of liver enzymes used for preparation S9 used in this study was 25 July 2016.

PREPARATION OF RAT LIVER HOMOGENATE S9 FRACTION
- On Day 4, the rats were euthanised and the livers were removed aseptically using sterile surgical tools.
- After excision, livers were weighed and washed several times in 0.15 M KCl.
- The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenised.
- Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained.
- The freshly prepared S9 fraction was aliquoted into 1-3 mL portions, frozen quickly and stored at -80 ± 10 °C.
- The date of preparation of S9 fraction for this study was 28 July 2016 (CiToxLAB code: E12440).
- The sterility of the preparation was confirmed. The protein concentration of the preparation was determined by a chemical analyser at 540 nm in the Clinical Chemistry Laboratory of CiToxLAB Hungary Ltd.
- The mean protein concentration of the S9 fraction used was determined to be 29.2 g/L.
- The biological activity in the Salmonella assay of S9 was characterized using the two mutagens (2-Aminoanthracene and Benzo(a)pyrene) that requires metabolic activation by microsomal enzymes.
- The batch of S9 used in this study functioned appropriately.

S9 MIX
- Salt solution for the S9 mix contained NADP Na (7.66 g); D-glucose-6-phosphate Na (3.53 g); MgCl2.6H20 (4.07 g); KCl (6.15 g); sufficient distilled water to give 1000 mL. Sterilisation was performed by filtration through a 0.22 µm membrane filter.
- The complete S9 mix was freshly prepared and contained ice cold 0.2 M sodium phosphate buffer pH 7.4 (500 mL); S9 rat liver homogenate (100 mL); salt solution for S9 mix (400 mL).
- The S9 mix was kept in an ice bath prior to addition to the culture medium.
- Sodium phosphate buffer 0.2 M (pH 7.4) solution A contained Na2HPO4 (71.63 g) and sufficient distilled water to give a volume of 1000 mL. Sterilisation was performed in an autoclave at 121 °C.
- Sodium phosphate buffer 0.2 M (pH 7.4) solution B contained Na2PO4.H2O (27.6 g) and sufficient distilled water to give a volume of 1000 mL. Sterilisation was performed in an autoclave at 121 °C.
- Sodium phosphate buffer pH 7.4 contained solution A (880 mL) and solution B (120 mL).

TEST PROCEDURE
- The study included a preliminary compatibility test, a preliminary range finding test (informatory toxicity test), an initial mutation test, a confirmatory mutation test and a complementary confirmatory mutation test.
- The plate incorporation method was used.

CONCENTRATIONS
- Concentrations were selected on the basis of the preliminary compatibility test and preliminary range finding test (informatory toxicity test).
- The same concentrations were used in the Initial Mutation Test, Complementary Initial Mutation Test and Confirmatory Mutation Test.

PRELIMINARY COMPATIBILITY TEST
- Solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO), N,N-Dimethylformamide (DMF) and Acetone. The test item was insoluble in Distilled water and in DMSO (after 2-minutes incubation ultrasonic water bath) at 100, 50 and 33.3 mg/mL concentration. Partial dissolution was observed at 100, 50 and 33.3 mg/mL (precipitate was observed) concentration using DMF (after 2-minutes
incubation ultrasonic water bath). Partial dissolution was observed at 100, 50 and 33.3 mg/mL (quickly settling formulation) concentration using Acetone and the formulation at 33.3 mg/mL concentration using Acetone as vehicle (after 2-minutes incubation in an ultrasonic water bath) was a homogeneous formulation.
- Note: The test item was not a homogeneous formulation at 100 and 50 mg/mL concentration after 2-
minutes incubation ultrasonic water bath.
- Based on the results, Acetone was selected as vehicle (solvent) for the study. The obtained stock formulation (150 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension to examine the formulation compatibility (Preliminary Compatibility Test).

PRELIMINARY CONCENTRATION RANGE-FINDING TEST (INFORMATORY TOXICITY TEST)
- Based on the available information and the solubility and compatibility test, 33.33 mg/mL stock solution was prepared in Acetone, which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item. In the Preliminary Concentration Range Finding Test the plate incorporation method was used.

TEST ITEM CONCENTRATIONS IN THE MUTAGENICITY TESTS
- Based on the results of the preliminary test, 33.33 mg/mL stock solution was prepared in Acetone, which was diluted by serial dilutions in six steps to obtain seven dosing formulations for lower doses. The maximum test concentration was 5000 μg test item/plate.
- Examined concentrations in the Initial Mutation Test, in the Confirmatory Mutation Test and in the Complementary Initial Mutation Test in Salmonella typhimurium TA1537 strain with and without metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81 and 5μg/plate.

CONTROL GROUPS USED IN THE TESTS
- Strain-specific positive and negative (vehicle/solvent) controls, both with and without metabolic activation were included in each test.
- In addition, untreated control was used demonstrating that the chosen vehicle (solvent) induced no deleterious or mutagenic effects.
- The control groups are summarised in Table 4 (below).
- If the solvent of the positive control substance differed from the vehicle (solvent) of the test item, both solvents were run in the assay.

EXPOSURE PROCEDURE
- A standard plate incorporation procedure was performed as an Initial Mutation Test, Complementary Initial Mutation Test and Confirmatory Mutation Test. Bacteria (cultured in Nutrient Broth No.2) were exposed to the
test item both in the presence and absence of an appropriate metabolic activation system.
- Note: The Confirmatory Mutation Test was performed using the plate incorporation method because the required treatment volume (150 μL) of Acetone as vehicle is more suitable for this method than the pre-incubation method.
- Molten top agar was prepared and kept at 45°C. The equivalent number of minimal glucose agar plates (three plates per concentration and for each control) was properly labelled. The test item and other components were prepared freshly and added to the overlay (45°C).
- Tubes contained top agar (2000 µL); vehicle (solvent) or test item solution (or reference controls) 150 (50 µL); overnight culture of test strain (100 µL); phosphate buffer (pH 7.4) or S9 mix (500 µL).
- Note: Treatment volume was 150 μL for test item formulation and its vehicle control in the main experiments; treatment volume was 50 μL for positive control substance formulations and their vehicle controls.
- This solution was mixed and poured on the surface of minimal agar plates. For activation studies, instead of phosphate buffer, 0.5 mL of the S9 mix was added to each overlay tube. The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (solvent) and positive controls.
- After preparation, the plates were incubated at 37 °C for 48 hours.

VALIDITY CRITERIA
- The number of revertant colonies of the negative (vehicle/solvent) and positive controls were in the historical control range in all strains of the main tests.
- At least five analysable concentrations were presented in all strains of the main tests.

CRITERIA FOR A POSITIVE RESPONSE
- A dose–related increase in the number of revertants occurred and/or a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
- An increase was considered biologically relevant if:
(i) The number of reversions was more than two times higher than the reversion rate of the
negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains.
(ii) The number of reversions was more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
- According to the guidelines, statistical methods may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

CRITERIA FOR A NEGATIVE RESPONSE
- A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.

Evaluation criteria:

EVALUATION OF EXPERIMENTAL DATA
- Colony numbers on the untreated / negative (vehicle/solvent) / positive control and test item treated plates were determined by manual counting. Visual examination of the plates was also performed; precipitation or signs of growth inhibition (if any) were recorded and reported. The mean number of revertants per plate, the standard
deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.
- The Mutation factor (MF) = mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY COMPATIBILITY TEST
- Based on the available information and the results of the solubility testing, Acetone was selected as vehicle (solvent) of the study.
- The results of the Preliminary Compatibility Test are summarised in Table 6 (attached).

PRELIMINARY CONCENTRATION RANGE-FINDING TEST
- In the Preliminary Concentration Range Finding Test (Informatory Toxicity Test), the plate incorporation method was used. This test was performed using Salmonella typhimurium TA98 and TA100 strains in the presence and absence of metabolic activation system (± S9 mix) with appropriate untreated, negative (vehicle/solvent) and
positive controls. In the test, each sample (including the controls) was tested in triplicate.
- Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test.
- In the preliminary experiment, the numbers of revertant colonies were mostly in the normal range (differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system).
- Precipitate/slight precipitate was observed in the both tester strains with and without metabolic activation at the concentrations of 5000, 2500 and 1000μg/plate.
- No inhibitory, cytotoxic effect of the test item was observed in the Preliminary Concentration Range Finding Test.
- The experimental results (revertant colony numbers per plate, mutation factors and standard deviations) are detailed in Table 7 and in Appendix 3 (attached).

INITIAL AND CONFIRMATORY MUTATION TESTS
- In the Initial Mutation Test, Confirmatory Mutation Test and Complementary Mutation Test, the plate incorporation method was used.
- The Initial Mutation Test and Confirmatory Mutation Test were carried out using four Salmonella typhimurium strains (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2 uvrA strain in the presence and absence of a metabolic activation system (± S9 mix) with appropriate untreated, negative (vehicle/solvent) and
positive controls.
- The Complementary Initial Mutation Test was carried out using Salmonella typhimurium TA1537 strain in the presence and absence of a metabolic activation system (-S9 mix) with appropriate untreated, negative (vehicle/solvent) and positive controls. In the main tests, each sample (including the controls) was tested in triplicate.
- Note: In the Initial Mutation Test in Salmonella typhimurium TA1537 strain was infected. Therefore, an additional experiment (Complementary Initial Mutation Test) was performed in this strain in an additional experimental period (Experimental Period III) to complete the data. The experimental conditions were the same as in the Confirmatory Mutation Test. Results of the invalid experiment were not reported; however, all data are kept and archived in the raw data binder.
- Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test, in the Confirmatory Mutation Test and in the Complementary Initial Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.
- In the Initial Mutation Test and in the Complementary Initial Mutation Test (using the plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain at 50μg/plate concentration with metabolic activation (the observed mutation factor value was 1.50). However, there was no clear dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
- In the Confirmatory Mutation Test (plate incorporation method), the highest revertant rate was observed in Salmonella typhimurium TA98 at 15.81μg/plate concentration without metabolic activation (the observed mutation factor value was 1.30). However, there was no clear dose-response relationship, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
- Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main tests in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test.
- Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main tests at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological
variability of the test system.
- Precipitate/slight precipitate was detected on the plates in the Initial Mutation Test, in the Confirmatory Mutation Test and in the Complementary Initial Mutation Test in all examined strains with and without metabolic activation at 5000, 1581 and 500 μg/plate concentrations.
- Inhibitory, cytotoxic effect of the test item was not detected in the Initial Mutation Test, in the Confirmatory Mutation Test and in the Complementary Mutation Test.
- The experimental results (revertant colony numbers per plate, mutation factors, and standard deviations) are summarized in Tables 8-9 and Appendices 4-6 (attached)

VALIDITY OF THE TESTS
- Untreated, negative (vehicle/solvent) and positive controls were run concurrently. The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains.
- The examined concentration range was considered to be adequate as concentrations up to maximum recommended concentration (5000 μg/plate) were examined in the main tests. At least five analysable concentrations were presented in all strains with and without metabolic activation.
- The reference mutagens showed a distinct increase of induced revertant colonies in each strain with and without metabolic activation. The viability of the bacterial cells was checked by a plating experiment in each test. The study was considered to be valid.

Conclusions:

The experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test item demonstrated no mutagenic activity in the bacterial tester strains used in the study.

Executive summary:

GUIDELINE

The test item was investigated for potential mutagenic activity using the bacterial reverse mutation assay. The study was conducted in accordance with the Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No 471, "Bacterial Reverse Mutation Test" (21 July 1997), EPA Health Effects Test Guidelines, OPPTS 870.5100 "Bacterial Reverse Mutation Test", EPA 712-C-98-247 (August 1998) and Commission Regulation (EC) No. 440/2008, B.13/14. "Mutagenicity: Reverse Mutation Test Using Bacteria" (30 May 2008). The method also conformed to conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF.

 

METHODS

Experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

 

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Plate Incorporation Method) and a Complementary Mutation Test (Plate Incorporation Method).

 

Based on the results of a solubility tests, the test item was formulated in Acetone. Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test, in the Confirmatory Mutation Test and in the Complementary Initial Mutation Test in Salmonella typhimurium TA1537 strain with and without metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81 and 5μg/plate.

 

RESULTS

In the Initial Mutation Test, Confirmatory Mutation Tests and the Complementary Initial Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle control and were within the normal biological variability of the test system.

 

Inhibitory, cytotoxic effects of the test item were not detected in the Preliminary Concentration Range Finding Test and in the main tests in all examined strains with and without metabolic activation.

 

Precipitate/slight precipitate was detected on the plates in the Preliminary Concentration Range Finding Test, in the Initial Mutation Test, Confirmatory Mutation Test in all examined bacterial strains with and without metabolic activation at several concentrations.

 

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

 

CONCLUSION

The experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test item demonstrated no mutagenic activity in the bacterial tester strains used in the study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 March 2017 to 11 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

difference in treatment time during preliminary toxicity test with no impact on integrity or validity of the result (see below)

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

difference in treatment time during preliminary toxicity test with no impact on integrity or validity of the result (see below)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: chromosome aberration

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cells for this study were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1 % (v/v) Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10 % (v/v) heat-inactivated fetal bovine serum (DMEM-10, culture medium).

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital / β-naphthoflavone induced S9 mix

Test concentrations with justification for top dose:

- Assay 1 (3-hr treatment without S9 Mix, harvesting 20 hours from the beginning of treatment): 1000, 333.3, 111.1, 37.04 and 12.35 μg/mL.
- Assay 1 (3-hr treatment with S9 Mix, harvesting 20 hours from the beginning of treatment): 1000, 333.3, 111.1, 37.04 and 12.35 μg/mL.
- Assay 2 (20-hr treatment without S9 Mix, harvesting 20 hours from the beginning of treatment): 1000, 333.3, 111.1, 37.04 and 12.35 μg/mL.
- Assay 2 (3-hr treatment with S9 Mix, harvesting 20 hours from the beginning of treatment): 1000, 333.3, 111.1, 37.04 and 12.35 μg/mL.

Vehicle / solvent:

Acetone

Untreated negative controls:

yes

Remarks:

acetone (final concentration of 10 μL/mL)

Remarks:

Supplier VWR; Lot number 15J060514; Expiry date 31 October 2020; Room temperature storage conditions

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

ethylmethanesulphonate

Remarks:

EMS (Sigma Aldrich; Lot number BCBN1209V; Expiry date 31 May 2017; Room temperature storage under nitrogen)

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

CP (Sigma Aldrich; Lot number MKBX1822V; Expiry date 31 December 2018; Storage conditions 2-8 °C)

Details on test system and experimental conditions:

ARCHIVE SAMPLING
- An adequate sample of the test item was collected and retained under the same storage conditions as applied in the study in the Archives of CiToxLAB Hungary Ltd.

FORMULATION
- Stock formulations of the test item (100 mg/mL) were prepared as follows.
- The necessary amount of test item was weighed into a calibrated volumetric flask.
- The required volume of the vehicle (Acetone) was added and the formulation was incubated
mixed until homogeneity was reached.
- From the stock formulation, several dilutions were prepared using the selected vehicle to prepare dosing formulations.
- Sterile vehicle was used for formulation.
- The dosing formulations were prepared immediately before the treatment of the cells in a sterile hood.
- Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and short period of study.

NEGATIVE CONTROL
- Negative (vehicle) control samples were run concurrently with treatment groups.
- Based on the available data (CiToxLAB study code: 16/316-033EL), Acetone as vehicle was
suitable for the test.
- In each case, sterile vehicle was prepared (filtered sterile using a 0.22 μm syringe filter before use (Supplier: Millipore, Lot No.: R5SA78859 / R6NA19212, Expiry date: January 2019 / October 2019) and used for formulation.

POSITIVE CONTROLS
- Without metabolic activation: Ethyl methanesulfonate, a known mutagen and clastogen, was dissolved in DMEM and was used as a positive control for the non-activation experiments at a final concentration of 0.4 μL/mL or 1.0 μL/mL.
- With metabolic activation: Cyclophosphamide monohydrate, a clastogenic agent that requires metabolic transformation by microsomal enzymes, was dissolved in sterile physiological saline solution (0.9% NaCl infusion) for treatment and was used as a positive control item for the activation experiments at a final concentration of 6.0 μg/mL
- Positive control solutions were prepared immediately before the treatment of the cells and filtered sterile using a 0.22 μm syringe filter before use (Supplier: Millipore, Lot Number: R6DA42546, Expiry date: April 2019 was used in Assay 1 and Lot Number: R6KA05144, Expiry date: August 2019 was used in Assay 2).

INDICATOR CELLS
- V79: Chinese hamster lung, male
- ECACC Cat. No.: 86041102
- Lot No.: 10H016
- Date of working lot: 31 July 2015
- Supplier: ECACC (European Collection of Cells Cultures)
- Morphology: Fibroblast
- The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes it suitable for genetic toxicity assays with low background aberrations. These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12-14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks were kept in a freezer at -80 ± 10 °C (for short-term storage) or in liquid nitrogen (long-term storage). The stock was checked for mycoplasma infection. No infection of mycoplasma was noted.
- Trypsin-EDTA (0.25% Trypsin, 1mM EDTA) solution was used for cell detachment to subculture (cells were rinsed with 1X PBS before detachment). The laboratory cultures were maintained in 150 cm2 plastic flasks at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air. The V79 cells for this study were grown in Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine, 1% (v/v) Antibiotic-antimycotic solution (standard content: 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and 10% (v/v) heat-inactivated fetal bovine serum (DMEM-10, culture medium). When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study). During the treatments, the serum content of the medium was reduced to 5% (v/v) (DMEM-5).

EXTERNAL METABOLIC ACTIVATION SYSTEM
- An advantage of using in vitro cell cultures is the accurate control of the concentration and exposure time of cells to the test item under the study. However, due to the limited capacity of cells growing in vitro for metabolic activation of potential mutagens, an exogenous metabolic activation system is necessary.
- Many substances only develop mutagenic potential after they are metabolised. Metabolic activation of substances can be achieved by supplementing the cell cultures with liver microsome preparations (S9 mix).
- In the experiments with metabolic activation in this study, a cofactor-supplemented post-mitochondrial S9 fraction prepared from activated rat liver was used as an appropriate metabolic activation system.
- The post-mitochondrial fraction (S9 fraction) was prepared by the Microbiological Laboratory of CiToxLAB Hungary Ltd. according to Ames et al. and Maron and Ames. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived yearly.
- The supplier, batch number and expiry date of the chemicals used are described in the attached table.

INDUCTION OF RAT LIVER ENZYMES
- Male Wistar rats (345-441 g, animals were 9 weeks old at the initiation of E12440, 389-479 g, animals were 14-17 weeks old at the initiation of E12590) were treated with Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before sacrifice when food was removed. Initiation dates of the induction of liver enzymes used for preparation S9 used in this study was 25 July 2016 (CiToxLAB code: E12440) and 16 January 2017 (CiToxLAB code: E12590).

PREPARATION OF RAT LIVER HOMOGENATE S9 FRACTION
- On Day 4, the rats were euthanized (sacrifice was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels) and the livers were removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenised.
- Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was aliquoted into 1-3 mL portions, frozen quickly and stored at -80 ± 10ºC. The date of preparation of S9 fraction for these studies was 28 July 2016 (CiToxLAB code: E12440) and 19 January 2017 (CiToxLAB code: E12590).
- The protein concentration of the preparation was determined by a chemical analyser at 540 nm in the Clinical Chemistry Laboratory of CiToxLAB Hungary Ltd. The protein concentration of the S9 fraction used in the study was determined to be 29.2 g/L (CiToxLAB code: E12440) and 29.6 g/L (CiToxLAB code: E12590). The sterility of the preparation was confirmed in each case.
- The biological activity in the Salmonella assay of S9 was characterized using the two mutagens 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batches of S9 used in this study functioned appropriately.

PREPARATION OF S9 MIX
- The complete S9-mix was freshly prepared on the day of use contained S9 fraction (3 mL); HEPES 20 mM (2 mL); KCl 330 mM (1 mL); MgCl2 50 mM (1 mL); NADP 40 mM (1 mL); Glucose-6-phosphate 50 mM (1 mL); DME medium (1 mL).
- Prior to addition to the culture medium the S9-mix was kept in an ice bath.
- For all cultures treated in the presence of S9-mix, a 0.5 mL aliquot of the mix was added to each cell culture (final volume: 10 mL). The final concentration of the liver homogenate in the test system was 1.5%.

TOXICITY AND CONCENTRATION SELECTION
- Treatment concentrations for the mutation assay were selected based on the results of a short preliminary test.
- In this Preliminary Toxicity Test, two separate assays were performed. In Assay A, cells were treated for 3-hours in the presence and absence of S9-mix with a 20-hour harvesting time. In Assay B, cells were treated for 20 hours in the absence of S9-mix with a 20-hour harvesting time.
- The assays were performed with a range of test item concentrations to determine cytotoxicity. Treatment was performed as described for the main test. However, single cultures were used and positive controls were not included. Visual examination of the final culture medium was conducted at the beginning and end of the treatments. Measurement of pH and osmolality was also performed at the end of the treatment period.
- At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as RICC (Relative Increase in Cell Counts). Tabulated results of the preliminary experiments are given in Appendix 2 (attached).

CHROMOSOME ABERRATION ASSAYS
- The Chromosome Aberration Assays were conducted as two independent experiments (Assay 1 and Assay 2) in the presence and in the absence of metabolic activation. In Assay 1, 3-hour treatment was performed with and without metabolic activation (in the presence and absence of S9 mix); cells were harvested 20-hour after the beginning of the treatment. In Assay 2, a 3-hour treatment was performed with metabolic activation (in the presence of S9 mix) and 20-hour without metabolic activation (in the absence of S9 mix) in duplicate cultures; cells were harvested 20-hour after the beginning of the treatment.
- The negative (vehicle) control, positive control and 3 dose levels (based on the observed solubility and/or cytotoxicity results) were selected for chromosomal aberration analysis.

TREATMENT OF CELLS
- For the cytogenetic experiments, 1-3 day old cultures (more than 50 % confluency) were used. Cells were seeded into 92 x 17 mm tissue culture dishes at 5 x 10E05 cells/dish concentration and incubated for approximately 24 hours at 37 °C in 10 mL of culture medium (DMEM-10). Duplicate cultures were used for each test item concentration or controls.
- After the seeding period, the medium was replaced with 9.9 mL treatment medium (DMEM-5) in case of experiments without metabolic activation or with 9.4 mL treatment medium (DMEM-5) + 0.5 mL S9-mix in case of experiments with metabolic activation.
- Cells were treated with different concentration test item solutions, negative (vehicle) or positive control solution (treatment volume: 100 μL/dish in all cases) for the given period of time at 37 °C in the absence or presence of S9-mix. After the exposure period, the cultures were washed with DMEM-0 medium (Dulbecco’s Modified Eagle’s Medium supplemented with 2 mM L-glutamine and 1% (v/v) Antibiotic-antimycotic solution). Then, 10 mL of fresh culture medium were added into the dishes and cells were incubated further until the scheduled harvesting time.
- Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles) from the beginning of treatment.
- Solubility of the test item in the final treatment medium was visually examined at the beginning and end of the treatment in each case. Measurement of pH and osmolality was also performed at the end of the treatment period in both main tests.
- For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (vehicle) control as RICC (Relative Increase in Cell Counts).

PREPARATION OF CHROMOSOMES
- At 2 to 2.5 hours prior to harvesting, cell cultures were treated with Colchicine (0.2 μg/mL). The cells were swollen with 0.075 M KCl hypotonic solution for 4 minutes, then were washed in fixative (Methanol : Acetic-acid 3 : 1 (v : v) mixture) until the preparation became plasma free (4 washes). Then, a suspension of the fixed cells was dropped onto clean microscope slides and air-dried. The slides were stained with 5 % Giemsa solution, air-dried and coverslips were mounted. At least three slides were prepared for each culture.
- Note: Fixed cells were stored frozen in case if any additional slide dropping was required (as documented in the raw data and reported). After the finalization of the report, the remaining frozen cell suspension samples will be discarded.

EXAMINATION OF SLIDES
- The stained slides were given random unique code numbers at the Test Facility by a person who was not involved in the metaphase analysis.
- The code labels covered all unique identification markings on the slides to ensure that they were scored without bias.
- The metaphase analysis (reference number of the Test Site is 77769 for this study) was conducted under the control of the Principal Investigator (CiToxLAB Scantox A/S, Denmark) in compliance with the OECD Principles of Good Laboratory Practice (as revised in 1997) and the Test Sites relevant SOPs. These Principles are in conformity with other international GLP regulations.
- At least 150 metaphases with 22±2 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
- Aberrations were defined in the following way:
(i) Gap: small unstained lesion smaller than the width of a chromatid and with minimal misalignment of the chromatid(s)
(ii) Break: unstained lesion larger than the width of a chromatid, or with clear misalignment
(iii) Exchange: breakage and reunion of chromatids within a chromosome, or between chromosomes
(iv) Chromatid-type: structural chromosome damage expressed as breakage of single chromatids or breakage and reunion between chromatids
(v) Chromosome-type: structural chromosome damage expressed as breakage, or breakage and reunion, of both chromatids at an identical site.
- Fragments could arise from breakage and exchange events. When the origin of a fragment was clear, it was recorded under that category (e.g. a dicentric chromosome with a fragment was recorded as one chromosome exchange event). When the origin of the fragment was not clear, it was recorded as a chromatid break. Metaphases with more than five aberrations (excluding gaps) were recorded as showing multiple damage. The examination of slides from a culture may be halted when 25 or more metaphases with aberrations (excluding gaps) have been recorded for that culture.
- Additionally, the number of polyploid and endoreduplicated cells was scored. Polyploid metaphases are defined as metaphases with approximate multiples of the haploid chromosome number (n), other than the diploid number (i.e. ca. 3n, 4n etc). Endoreduplicated metaphases have chromosomes with 4, 8, etc. chromatids. Marked reductions in the numbers of cells on the slides were recorded if needed.
- The Vernier co-ordinates of at least five metaphases (with aberrations, where possible) were recorded for each culture.
- When the metaphase analysis has been completed for each test, the slide code was broken and the number of metaphases with aberrations (excluding gaps) and the types of aberrations for each culture were presented in tables.
- When the metaphase analysis has been performed, the original raw data (record sheets) for the metaphase analysis and the microscope slides are shipped back to the Test Facility (CiToxLAB Hungary Ltd). After the slide reading phase of the study had ended, the Principal Investigator issued a Work Phase Report.

Rationale for test conditions:

INTRODUCTION
- Chromosome aberration tests, which detect structural chromosome aberrations in somatic and/or germ cells play an important role in the evaluation of genotoxicity of a given test item. Structural aberrations develop due to breaks in one or both DNA strands, which can result in chromosome fragments (breaks, deletions). Faulty reunion of chromosome fragments results in formation of exchanges. These aberrations can be detected and quantified using light microscope. Extensive chromosome breaks usually cause cell death; small changes (breaks, deletions, translocations, inversions etc.) are, however, not necessarily lethal and can be regarded as an indication of molecular events, which might lead to malignant cell transformation.

OBJECTIVE
- The objective of this study was to determine whether the test item could induce structural chromosome aberrations in cultured Chinese hamster cells.

Evaluation criteria:

EVALUATION OF RESULTS
- The assay is considered valid, if the following criteria are met:
(i) The negative (vehicle) control data are within the laboratory’s normal range for the spontaneous aberration frequency.
(ii) The positive controls induce increases in the aberration frequency, which are significant.
- The test item is considered to have shown clastogenic activity in this study if all of the following criteria are met:
(a) Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
(b) The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
(c) The increases are statistically significant.
(d) The increases are not associated with large changes in pH or osmolarity of the treated cultures.
- The historical control data for this laboratory were also considered in the evaluation. Evidence of a dose-response relationship (if any) was considered to support the conclusion.
- The test item is concluded to have given a negative response if no reproducible, statistically significant increases are observed.
- Historical control data are shown in Appendix 5 (attached).

Statistics:

- For statistical analysis, Fisher’s exact test was used. The parameter evaluated for statistical analysis was the number of cells with one or more chromosomal aberrations excluding gaps.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

VEHICLE AND CONCENTRATION SELECTION
- Based on the available information (Material Safety Data Sheet), the test item is practically insoluble in water. Based on the result of a short solubility test which was performed in the 16/316-033EL study, the test item was insoluble at 200 mg/mL concentration using Acetone, Dimethyl sulfoxide (DMSO) and N,NDimethylformamide (DMF); but the formulation at 100 mg/mL concentration using Acetone as vehicle was suitable for the test (after approx. 20-30 minutes of vigorous mixing by magnetic stirrer and ultrasound water bath) while the other vehicles did not result in proper formulation at the same concentration. Therefore, Acetone was selected as the vehicle of the study. The highest examined concentration in the preliminary test was 1000 μg/mL.
- Two Concentration Selection Cytotoxicity Assays (Assay A: 3-hour treatment with and without metabolic activation, 20-hour harvesting time; and Assay B: 20-hour treatment without metabolic activation, 20-hour harvesting time) were performed as part of the study to establish an appropriate concentration range for the Chromosome Aberration Assays, both in the absence and in the presence of a metabolic activation system.
- A total of ten test concentrations between 1000 and 1.953 μg/mL were used to evaluate toxicity in the presence and absence of metabolic activation in each cytotoxicity assay. Detailed results of the cytotoxicity assays are presented in Tables 2, 3 and 4 of Appendix 2 (attached). Treatment concentrations for the chromosome aberration assays were selected on the basis of results of the performed Concentration Selection Cytotoxicity Assays according to the OECD guideline instructions.

CHROMOSOME ABERRATION ASSAYS
- In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 1000, 333.3, 111.1, 37.04 and 12.35 μg/mL (experiment with and without metabolic activation).
- In Assay 1, insolubility (precipitate / minimal amount of precipitate) was detected at the beginning and at the end of the treatment period in the final treatment medium in the 1000-111.1 μg/mL concentration range with and without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in any samples of this assay (for more details see Table 5 and Table 6 of Appendix 3, attached). Therefore, concentrations of 333.3, 111.1 and 37.04 μg/mL (a total of three) were chosen for evaluation in the experiment with and without metabolic activation.
- In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 1000, 333.3, 111.1, 37.04 and 12.35 μg/mL (experiment with and without metabolic activation).
- In Assay 2, similarly to the first experiment, insolubility (precipitate / minimal amount of precipitate) was detected at the beginning and at the end of the treatment period in the final treatment medium in the 1000-111.1 μg/mL concentration range without metabolic activation and in the 1000-37.04 μg/mL concentration range with metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in any samples of this assay (for more details see in Tables 7 and 8 of Appendix 3, attached). Concentrations of 333.3, 111.1 and 37.04 μg/mL (a total of three) were evaluated in the experiment without metabolic activation, and concentrations of 111.1, 37.04 and 12.35 μg/mL (a total of three) were evaluated in the experiment with metabolic activation.
- None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values. Summarised data are shown in Tables 9-10 of Appendix 4 (attached).
- Polyploid metaphases (1) were found in some cases in the negative (vehicle) control or test item treated samples in the performed experiments, but their incidence was not related to treatment with the test item. No endoreduplicated metaphases were detected in the performed experiments.

VALIDITY OF THE STUDY
- The tested concentrations in the chromosome aberration assays were selected based on the results of the preliminary experiments. Insolubility was detected in all experiments with and without metabolic activation; while cytotoxicity was not detected in any experiment with and without metabolic activation. The evaluated concentration ranges of Assay 1 and Assay 2 were considered to be adequate, as they covered the range from toxicity to no or little toxicity (experiments with long treatment without metabolic activation) or they covered the range from insolubility to little or no insolubility (experiment with short treatment with and without metabolic activation).
- Three test item concentrations were examined in each experiment.
- Historical control data are presented in Appendix 5 (attached). The spontaneous aberration frequencies of the negative (vehicle) controls in the performed experiments were within the general historical control range of the testing laboratory.
- In the performed experiments, the positive control substances (Cyclophosphamide (CP) in the experiments with metabolic activation and Ethyl methanesulfonate (EMS) in the experiments without metabolic activation) caused the expected statistically significant increase in the number of cells with structural chromosome aberrations (see Tables 9-10 of Appendix 4, attached) demonstrating the sensitivity of the test system in each assay.
- The study was considered to be valid.

Conclusions:

The test item did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, the test item was not considered clastogenic in this test system.

Executive summary:

GUIDELINE

Structural chromosomal aberrations were investigated in cultured mammalian cells in accordance with OECD Guidelines for Testing of Chemicals, Section 4, No. 473, “In Vitro Mammalian Chromosome Aberration Test”, 29 July 2016 and Commission Regulation (EU) 2017/735 of 14 February 2017 B.10. "Mutagenicity – In Vitro Mammalian Chromosome Aberration Test".

 

METHODS

The test material was tested in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in Acetone and it was examined up to cytotoxic concentrations and/or solubility limit according to the OECD guideline recommendations. In the performed independent Chromosome Aberration Assays using duplicate cultures at least 300 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each test item treated, negative (vehicle) and positive control sample.

 

In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 1000, 333.3, 111.1, 37.04 and 12.35 μg/mL (experiment with and without metabolic activation).

 

In Assay 1, insolubility (precipitate / minimal amount of precipitate) was detected at the beginning and at the end of the treatment period in the final treatment medium in the 1000-111.1 μg/mL concentration range with and without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 333.3, 111.1 and 37.04 μg/mL (a total of three) were chosen for evaluation in the experiment with and without metabolic activation.

 

In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 1000, 333.3, 111.1, 37.04 and 12.35 μg/mL (experiment with and without metabolic activation).

 

In Assay 2, similarly to the first experiment, insolubility (precipitate / minimal amount of precipitate) was detected at the beginning and at the end of the treatment period in the final treatment medium in the 1000 to 111.1 μg/mL concentration range without metabolic activation and in the 1000 to 37.04 μg/mL concentration range with metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in any samples of this assay. Concentrations of 333.3, 111.1 and 37.04 μg/mL (a total of three) were evaluated in the experiment without metabolic activation, and concentrations of 111.1, 37.04 and 12.35 μg/mL (a total of three) were evaluated in the experiment with metabolic activation.

 

RESULTS

None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.

 

Polyploid metaphases (1) were found in some cases in the negative (vehicle) control or test item treated samples in the performed experiments, but their incidence was not related to treatment with the test item. No endoreduplicated metaphases were detected in the performed experiments.

 

The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.

 

CONCLUSION

The test item did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, the test item was not considered clastogenic in this test system.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 September 2016 to 16 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Remarks:

3.7.2C

Details on mammalian cell type (if applicable):

Product No.: CRL-9518
Lot No.: 1661603
Supplier: American Type Culture Collection (Manassas,Virginia, USA)
Date of receipt: 22 January 2004
Date of working lot: 21 January 2016

Metabolic activation:

with and without

Metabolic activation system:

Rat liver homogenate S9 fraction induced with phenobarbital and β-naphthoflavone

Test concentrations with justification for top dose:

- Assay 1: 3-hour treatment with metabolic activation: 1000, 333.33, 111.11, 37.04, 12.35 and 4.12 μg/mL
- Assay 1: 3-hour treatment without metabolic activation: 1000, 333.33, 111.11, 37.04, 12.35 and 4.12 μg/mL
- Assay 2: 3-hour treatment with metabolic activation: 1000, 333.33, 111.11, 37.04, 12.35 and 4.12 μg/mL
- Assay 2: 24-hour treatment without metabolic activation: 1000, 333.33, 111.11, 37.04, 12.35 and 4.12 μg/mL

Vehicle / solvent:

Acetone

Negative solvent / vehicle controls:

yes

Remarks:

acetone

Remarks:

VWR; Lot number 15J060514; Expiry date 31 October 2020; Room temperature storage conditions

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide anhydrous (DMSO)

Remarks:

Sigma Aldrich; Lot number SZBG1310V; Expiry date 25 April 2019; Room temperature storage under nitrogen

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

NQO (Sigma Aldrich; Lot number WXBC1554V; Expiry date 31 October 2017; Storage conditions -23 plus or minus 8 °C)

Positive controls:

yes

Remarks:

with metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

CP (Sigma Aldrich; Lot number MKBX1822V; Expiry date 31 December 2018; Refrigerated storage at 2-8 °C)

Details on test system and experimental conditions:

OBJECTIVE OF THE STUDY
- The objective of this study was to determine whether the test item induces genotoxicity (point mutations and/or gross chromosomal changes) at the thymidine kinase (tk) locus in L5178Y 3.7.2 C mouse lymphoma cells cultured in vitro in the absence and presence of a rat liver metabolising system.

FORMULATION
- Based on the available information (Material Safety Data Sheet), the test item was practically insoluble in water. Bases on the result of a short solubility test, the test item was insoluble at 200 mg/mL concentration using Acetone, Dimethyl sulfoxide (DMSO), N,N-Dimethylformamide (DMF) and 1% methyl cellulose solution (1% MC); but the formulation at 100 mg/mL concentration using Acetone as vehicle was suitable for the test (after approx. 20-30 minutes of vigorous mixing by magnetic stirrer and ultrasound water bath), while other vehicle did not results in proper formulation at the same concentration. Therefore, Acetone was selected as vehicle of the study. The selected vehicle was compatible with the survival of the cells and the S9 activity.
- For the treatments in the study, stock formulations (100 mg/mL) were prepared in the Pharmacy of the Test Facility as follows. The necessary amount of test item was weighed into a calibrated volumetric flask; the required volume of the vehicle (solvent) was added and the mixture was vigorously stirred by a magnetic stirrer for approximately 20-30 minutes to obtain a homogenous formulation (incubation in an ultrasound water bath was also used, if needed, for proper dissolution). The stock formulation was transferred to the testing laboratory where several dilutions were prepared in a sterile hood using the selected vehicle (solvent) for dosing formulations. In each case, the vehicle was filtered sterile using a 0.22 μm syringe filter (Supplier:
Millipore, Lot No.: R5SA78859, Expiry date: January 2019) before the preparation of the dosing formulations. The stock formulations and all the dilutions were prepared immediately before the treatment of the cells. No purity conversion was applied in the study as agreed by the Sponsor.
- Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

ARCHIVE SAMPLING
- An adequate sample of the test item was collected and retained under the same storage conditions as used during the study in the Archives of CiToxLAB Hungary Ltd.

POSITIVE AND NEGATIVE CONTROLS
- Negative (vehicle) control: Negative (vehicle) control cultures were treated with the selected vehicle (solvent) alone in the same way as the test item treated cultures. In addition, untreated control sample was also used in each assay to demonstrate that the selected vehicle had no mutagenic effects. Dimethyl sulfoxide was used for vehicle (solvent) of the positive control chemicals.
- Positive control in the absence of metabolic activation: 4-Nitroquinoline-N-oxide, a widely used positive control in the absence of metabolic activation (selected based on the scientific literature, the experience of the Test Facility and the availability of historical control data), was dissolved in Dimethyl sulfoxide (DMSO) to form a stock solution at 20 μg/mL or 10 μg/mL and stored at -80 ±10 °C. Aliquots were removed from the ultrafreezer right before use, allowed to thaw and diluted with DMSO to 0.15 μg/mL (diluted from the 20 μg/mL stock solution) for 3-hour treatment and 0.10 μg/mL (diluted from the 10 μg/mL stock solution) for 24-hour treatment.
- Positive control in the presence of metabolic activation: Cyclophosphamide, a genotoxic agent that requires metabolic transformation by microsomal enzymes, was dissolved in DMSO right before use to prepare a 400 μg/mL stock solution. It was used in the experiments with metabolic activation at a final concentration of 4 μg/mL.
- Positive control solutions were freshly prepared at the beginning of the experiments in the testing laboratory and were filtered sterile using a 0.22 μm syringe filter before use (Supplier: Millipore, Lot No.: R6KA05144, Expiry date: August 2019).

TEST SYSTEM
- The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection. Cells were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/--mutants and checked for the absence of mycoplasma.
- For each experiment, one or more vials was thawed rapidly, cells were diluted in RPMI-10 medium and incubated at 37 ± 0.5 °C in a humidified atmosphere containing approximately 5% CO2 in air.
- When cells were growing well, subcultures were established in an appropriate number of flasks (after thawing, the cells were subcultured no more than 5 times before used in the study).

CHEMICALS USED IN THE EXPERIMENTS
- Chemicals used in the experiments are summarised in the attached table.

GROWTH MEDIA
- Three types of RPMI 1640 medium were prepared as shown in the table below.

EXTERNAL METABOLID ACTIVATION SYSTEM
- The post-mitochondrial fraction (S9 fraction) was prepared from rat liver by the Microbiological Laboratory of CiToxLAB Hungary Ltd. The documentation of the preparation of this post-mitochondrial fraction is stored in the reagent notebook in the Microbiological Laboratory which is archived annually.
- Induction of Liver Enzymes: Male Wistar rats (345-441 g, animals were 9 weeks old at the initiation) were treated with Phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage (for both inducers) for three consecutive days. Rats were given drinking water and food ad libitum until 12 hours before euthanasia when food was removed. Euthanasia was by ascending concentration of CO2, confirmed by cutting through major thoracic blood vessels. Initiation of the induction of liver enzymes used in the preparation of S9 fraction used in this study was 25 July 2016.
- Preparation of Rat Liver Homogenate S9 Fraction: On Day 4, the rats were euthanised and the livers removed aseptically using sterile surgical tools. After excision, livers were weighed and washed several times in 0.15 M
KCl. The washed livers were transferred to a beaker containing 3 mL of 0.15 M KCl per g of wet liver, and homogenized. Homogenates were centrifuged for 10 min at 9000 g and the supernatant was decanted and retained. The freshly prepared S9 fraction was distributed in 1-5 mL portions, frozen quickly and stored at -80 ± 10 °C. Sterility of the preparation was confirmed.
- The protein concentration was determined by colorimetric test by chemical analyser at 540 nm in the Clinical Chemistry Laboratory of CiToxLAB Hungary Ltd. The protein concentration of the S9 fraction used was determined to be 29.2 g/L. The date of preparation of S9 fraction for this study was 28 July 2016 (CiToxLAB code: E12440). The biological activity of each batch of S9 was characterized in the Salmonella assay using 2-Aminoanthracene and Benzo(a)pyrene, that requires metabolic activation by microsomal enzymes. The batch of S9 used in this study was found active under the test conditions.
- The S9-mix was prepared as shown in the table below.
- For all cultures treated in the presence of S9-mix, a 1 mL aliquot of the mix was added to each cell culture (19 mL) to give a total of 20 mL. The final concentration of the liver homogenate in the test system was 2 %. Cultures treated in the absence of S9-mix received 1 mL of 150 mM KCl (except for the 24-hour treatment). Prior to addition to the culture medium, the S9-mix was kept in an ice bath.

PRELIMINARY TOXICITY TEST
- A preliminary toxicity test was performed to select dose levels for the main assays. During the preliminary test, a 3-hour treatment in the presence and absence of S9-mix and a 24-hour treatment in the absence of S9-mix were performed with a range of test item concentration to determine toxicity.
- The procedures were performed as described for the main mutation assays; however, the test used single cultures and positive controls were not included. Following treatments, cell concentrations were determined using a haemocytometer. Visual examination for any kind of insolubility in the final culture medium was conducted at the beginning and end of treatment. Measurement of pH and osmolality was performed at the end of the
treatment period. Then cells were transferred for the expression period for two extra days and repeated cell counting was performed.

MAIN MUTATION ASSAYS
- In Assay 1, cells were treated for 3 hours in the presence and absence of S9 mix. In Assay 2, cells were treated for 3 hours in the presence of S9 mix and for 24 hours in the absence of S9-mix.
- A suitable volume (0.2 mL) of RPMI-5 medium, vehicle (solvent), test item formulations or positive control solutions, and 1.0 mL of S9-mix (in experiments with metabolic activation) or 1.0 mL of 150 mM KCl (in case of 3-hour treatment without metabolic activation) were added to a final volume of 20 mL per culture in each
experiment. For the 3-hour treatments, 107 cells were placed in each of a series of 75 cm2 sterile flasks. For the 24-hour treatment, 6x106 cells were placed in each of a series of 25 cm2 sterile flasks. The treatment medium contained a reduced serum level of 5% (v/v) RPMI-5.
- Duplicate cultures were used for each treatment. Cultures were visually examined at the beginning and end of treatments. During the treatment period, cultures were incubated at 37 ± 1 °C (approximately 5% CO2 in air). Gentle shaking was used during the 3-hour treatments. Measurement of pH and osmolality was also performed after the treatment period.
- Then cultures were centrifuged at 2000 rpm (approximately 836 g) for 5 minutes, washed with tissue culture medium and suspended in at least 20 mL RPMI-10. The number of viable cells in the individual samples was counted manually using a haemocytometer.
- Where sufficient cells survived, cell density was adjusted to a concentration of 2 x 10E05 cells/mL. Cells were transferred to flasks for growth through the expression period (maximum 30 mL of suspension) or diluted to be plated for survival.
- Concentrations in the main tests were as shown in the table below.

PLATING FOR SURVIVAL
- Cultures of cell density 2x10E05 cells/mL, were further diluted to 8 cells/mL as shown in the table below.
- Using a multi-channel pipette, 0.2 mL of the final concentration of each culture were placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks.
- Wells containing viable clones were identified by eye using background illumination and counted.

EXPRESSION PERIOD
- To allow expression of TK- mutations, cultures were maintained in flasks for 2 days. During the expression period, subculturing was performed daily. On each day, cell density was adjusted to a concentration of 2x105 cells/mL (whenever possible) and transferred to flasks for further growth.
- On completion of the expression period, six test item treated samples, untreated, negative (vehicle) and positive controls were plated for determination of viability and 5-trifluorothymidine (TFT) resistance.

PLATING FOR VIABILITY
- At the end of the expression period, the cell density in the selected cultures was determined and adjusted to 1 x 10E04 cells/mL with RPMI-20 for plating for a viability test. Samples from these cultures were diluted to 8 cells/mL as shown in the table below.
- Using a multi-channel pipette, 0.2 mL of the final concentration of each culture was placed into each well of two, 96-well microplates (192 wells) averaging 1.6 cells per well. Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for 12 days. Wells containing viable clones were identified by eye using background illumination and counted.

PLATING FOR -TRIFLUOROTHYMIDINE (TFT) RESISTANCE
- At the end of the expression period, the cell concentration was adjusted to 1 x 10E04 cells/Ml and TFT (300 μg/mL stock solution) was diluted 100-fold into these suspensions to give a final concentration of 3 μg/mL.
- Using a multi-channel pipette, 0.2 mL of each suspension was placed into each well of four, 96-well microplates (384 wells) at 2 x 10E03 cells per well.
- Microplates were incubated at 37 ºC ± 0.5 °C containing approximately 5% (v/v) CO2 in air for approximately two weeks (12 days) and wells containing clones were identified by eye and counted. In addition, scoring of large and small colonies was performed to obtain information on the possible mechanism of action of the test item, if any.

ANALYSIS OF RESULTS
- Methods associated with determination of survival or viability, calculation of Suspension Growth (SG) and Relative Total Growth (RTG) and determination of mutant frequency are described in the attached document.

ASSAY ACCEPTANCE CRITERIA
- The assay was considered valid if all of the following criteria were met (based on the relevant guidelines):
1. The mutant frequency in the negative (vehicle) control cultures fell within the normal range (50-170 mutants per 10E06 viable cells).
2. The positive controls met at least one of the following two criteria:
-The positive control chemical demonstrated an absolute increase in total MF that is, an increase above the spontaneous background MF of at least 300 x 10E-06. At least 40% of the IMF reflected in the small colony MF.
-The positive control substance had 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).
3. The plating efficiency (PEviability) of the negative (vehicle) controls was within the range of 65% to 120% at the end of the expression period.
4. At least four test concentrations were present, where the highest concentration produced approximately 80-90% toxicity (measured by RTG), resulted in precipitation, or it was 2 mg/mL, 2 μL/mL or 0.01 M (whichever is the lowest), or it is the highest practical (achievable) concentration. Note: When the test item is not of defined composition or (i.e. substance of unknown or variable composition, complex reaction product or biological material), the top concentration might need to be higher (e.g. 5 mg/mL) in the absence of sufficient cytotoxicity, to increase the concentration of each component.

Evaluation criteria:

EVALUATION CRITERIA
- The test item was considered to be clearly positive (mutagenic) in this assay if all the following criteria were met:
1. At least one concentration exhibited a statistically significant increase (p<0.05) compared with the concurrent negative control and the increase was biologically relevant (i.e. the mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle/solvent) control value).
2. The increases in mutation frequency were reproducible between replicate cultures and/or between tests (under the same treatment conditions).
3. The increase was concentration-related (p < 0.05) as indicated by the linear trend analysis.
- Results, which only partially satisfied the acceptance and evaluation criteria, were evaluated on a case-by-case basis and verified by an additional experiment documented by an amendment. Similarly, positive responses seen only at high levels of cytotoxicity might require careful interpretation when assessing their biological significance. The test item was considered clearly negative (non-mutagenic) in this assay if in all experimental conditions examined there was no concentration related response or, if there is an increase in MF, but it did not exceed the GEF. Then, test item was considered unable to induce mutations in this test system.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

3.7.2 C cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

PRELIMINARY EXPERIMENT
- Treatment concentrations for the mutation assay were selected based on the results of a short Preliminary Toxicity Test. 3-hour treatment in the presence and absence of metabolic activation system (S9-mix) and 24-hour treatment in the absence of metabolic activation system was performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest concentration tested in
the preliminary experiment using Acetone* as vehicle was 1000 μg/mL (the highest achievable concentration based on the limited solubility of the test item). Tabulated results of the preliminary experiment are given in Appendix 4 (attached).
- Based on the available information of another study performed at the Test Facility, the 200 mg/mL formulation was achievable using corn oil as vehicle. Therefore, the 2000 μg/mL concentration had been also tested in the preliminary experiment, but it was incompatible with the test system.
- Insolubility was detected in the preliminary experiments. Therefore, concentrations up to the solubility limit (and maximum achievable concentration) were selected for the main experiments according to the instructions of the relevant OECD guideline. Six concentrations were selected for the main experiments in each assay.

MUTATION ASSAYS
- In the mutation assays, cells were exposed to the test item for 3 hours with or without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). The cells were plated for determination of survival data and in parallel subcultured without test item for approximately 2 days to allow expression of the genetic changes. At the end of the expression period, cells were allowed to grow and form colonies for approximately 2 weeks (12 days) in culturing plates with and without selective agent (TFT) for determination of mutations and viability.
- In Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Treatment concentrations were 1000, 333.33, 111.11, 37.04, 12.35 and 4.12 μg/mL.
- Data are presented for survival (see Appendix 5, attached), viability (see Appendix 6, attached) and mutagenicity (see Appendix 7, attached). Results of the visual examination after treatment, as well as pH and osmolality data are presented in Appendix 16 (attached).
- In Assay 1, no large changes in pH or osmolality were detected in the final treatment medium at the end of the treatment. Insolubility was detected in the final treatment medium at the end of the treatment in the 37.04 - 1000 μg/mL concentration range (experiment with metabolic activation) and 12.35 - 1000 μg/mL concentration range (experiment without metabolic activation).
- In the presence of S9-mix (3-hour treatment), no cytotoxicity of the test item was observed (survival results are shown in Table 1 of Appendix 5, attached). An evaluation was made using data of the highest concentration of 1000 μg/mL and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase was seen at any examined concentrations. No significant dose response to the treatment was indicated by the linear trend analysis (for more details see Table 9 of Appendix 7, attached). Therefore, this experiment was considered as being negative.
- In the absence of S9-mix (3-hour treatment), no cytotoxicity of the test item was observed (survival results are detailed in Table 2 of Appendix 5, attached). An evaluation was made using data of the highest concentration of 1000 μg/mL and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined
concentrations (for details see Table 10 of Appendix 7, attached). No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment was considered as being negative.
- In Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 24-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Treatment concentrations were 1000, 333.33, 111.11, 37.04, 12.35 and 4.12 μg/mL.
- Data of Assay 2 are presented for survival (see Appendix 5, attached), viability (see Appendix 6, attached) and mutagenicity (see Appendix 7, attached). Results of the visual examination after treatment, as well as pH and osmolality data are presented in Appendix 16 (attached).
- In Assay 2, no large changes in pH or osmolality were detected in the final treatment medium at the end of the treatment. Insolubility (precipitate / minimal amount of precipitate, in some cases oily film layer or minimal oily film layer was also detected) was detected in the final treatment medium at the end of the treatment in the 37.04 - 1000 μg/mL concentration range (experiments with and without metabolic activation).
- In the presence of S9-mix (3-hour treatment), similarly to the first test, no cytotoxicity of the test item was observed as are shown in Table 3 of Appendix 5 (attached). An evaluation was made using data of the highest concentration of 1000 μg/mL and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase was seen at any evaluated concentrations. No dose-response to the
treatment was indicated by the linear trend analysis (for more details see Table 11 of Appendix 7, attached). This result was considered as confirming the negative effect observed in the first main test in the experiment with metabolic activation.
- In the absence of S9-mix (24-hour treatment), no cytotoxicity was observed at any test item treated concentrations (survival results are shown in Table 4 of Appendix 5, attached). An evaluation was made using data of the highest concentration of 1000 μg/mL and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increases in the mutation frequency were seen at any evaluated concentrations. No dose-response to the treatment was indicated by the linear trend analysis (details are shown in Table 12 of Appendix 7, attached). This experiment confirmed the negative results seen in the first main test in the experiment without metabolic activation.
- Other minor increases in the mutation frequency were observed sporadically in Assays 1 and 2; however, they were without any statistical significance and the difference between the observed values and the relevant solvent control value did not exceed the global evaluation factor, so they were considered as biologically not relevant increases, just showing the biological variability of the test system.

VALIDITY OF THE MUTATION ASSAYS
- Untreated, negative (vehicle) and positive controls were run concurrently in the study. The spontaneous mutation frequency of the negative (vehicle) and untreated controls were in the recommended range in all cases.
- The positive controls (Cyclophosphamide in the presence of metabolic activation and 4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the controls and were in accordance with historical data in all assays (for historical control data see Appendix 18, attached). All of the positive control samples in the performed experiments fulfilled at least one of the
relevant OECD criteria.
- The plating efficiencies for the negative (vehicle) control of the test item and untreated control samples at the end of the expression period (PEviability) were within the acceptable range (65-120%) in all assays (see Tables 5-8 of Appendix 6, attached) Note: Slightly lower value (60.9%) was recorded for the DMSO vehicle control of the positive control substance in Assay 1 without metabolic activation: However, as the other controls were in the acceptable range, this fact was considered to be acceptable and have no impact on the results of the study.
- The number of test concentrations evaluated was six in each case, which met the acceptance criteria of at least four evaluated concentrations.
- The tested concentration range in the study was considered to be adequate as concentrations up to the maximum available concentration (based on the limited solubility of the test item) were examined in the study and lower test concentrations were spaced by a factor of three. The examined concentration range covered the range from insolubility to no insolubility.
- Suspension growth value of the untreated and negative (vehicle) control were slightly lower than the recommended value (see Appendix 17, attached) in some cases in the experiments, however as all of the observed spontaneous mutation frequency values of the untreated and negative (vehicle) control samples were in the recommended range, this fact was considered to be acceptable and not to adversely affect the results of the study.
- The overall study was considered to be valid.

Conclusions:

No mutagenic effect of the test item was observed either in the presence or in the absence of metabolic activation system under the conditions of the Mouse Lymphoma Assay.

Executive summary:

GUIDELINE

The study followed the procedures indicated by OECD Guidelines for the Testing of Chemicals, Section 4, No. 490, "In Vitro Mammalian Cell Gene Mutation Test using the Thymidine Kinase Gene", 29 July 2016 and Commission Regulation (EC) No. 440/2008 of 30 May 2008, B.17. "Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test" (Official Journal L 142, 31/05/2008).

 

METHODS

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of test item to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

 

Acetone was used as vehicle of the test item in this study. The test item was examined up to 1000 μg/mL (the maximum achievable concentration due to the limited solubility of the test item) in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:

(i) Assay 1: 3-hour treatment with metabolic activation: 1000, 333.33, 111.11, 37.04, 12.35 and 4.12 μg/mL

(ii) Assay 1: 3-hour treatment without metabolic activation: 1000, 333.33, 111.11, 37.04, 12.35 and 4.12 μg/mL

(iii) Assay 2: 3-hour treatment with metabolic activation: 1000, 333.33, 111.11, 37.04, 12.35 and 4.12 μg/mL

(iv) Assay 2: 24-hour treatment without metabolic activation: 1000, 333.33, 111.11, 37.04, 12.35 and 4.12 μg/mL

 

RESULTS

In Assays 1-2, there were no large changes in pH or osmolality after treatment. Insolubility was detected in the final treatment medium at the end of the treatment in the 37.04 - 1000 μg/mL concentration range (treatment with metabolic activation and long treatment without metabolic activation) and 12.35 - 1000 μg/mL concentration range (short treatment without metabolic activation).

 

In Assay 1, following a 3-hour treatment with metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 1000 μg/mL and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase was seen at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. Therefore, this experiment was considered as being negative.

 

In Assay 1, following a 3-hour treatment without metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 1000 μg/mL and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose response to the treatment was indicated by the linear trend analysis. This experiment was considered as being negative.

 

In Assay 2, following a 3-hour treatment with metabolic activation, similarly to the first test, no cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 1000 μg/mL and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase was seen at any evaluated concentrations. No dose-response to the treatment was indicated by

the linear trend analysis. This result was considered as confirming the negative effect observed in the first main test in the experiment with metabolic activation.

 

In Assay 2, following a 24-hour treatment without metabolic activation, no cytotoxicity was observed at any test item treated concentrations. An evaluation was made using data of the highest concentration of 1000 μg/mL and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increases in the mutation frequency were seen at any evaluated concentrations. No dose-response to the treatment was indicated by the linear trend analysis. This experiment confirmed the negative results seen in the first main test in the experiment without metabolic activation.

 

The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.

 

CONCLUSION

No mutagenic effect of the test item was observed either in the presence or in the absence of metabolic activation system under the conditions of the Mouse Lymphoma Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro

Ames test

The test item was investigated for potential mutagenic activity using the bacterial reverse mutation assay. The study was conducted in accordance with the Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No 471, "Bacterial Reverse Mutation Test" (21 July 1997), EPA Health Effects Test Guidelines, OPPTS 870.5100 "Bacterial Reverse Mutation Test", EPA 712-C-98-247 (August 1998) and Commission Regulation (EC) No. 440/2008, B.13/14. "Mutagenicity: Reverse Mutation Test Using Bacteria" (30 May 2008). The method also conformed to conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF.

Experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone induced rats.

 

The study included a Preliminary Compatibility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method), a Confirmatory Mutation Test (Plate Incorporation Method) and a Complementary Mutation Test (Plate Incorporation Method).

 

Based on the results of a solubility tests, the test item was formulated in Acetone. Concentrations of 5000; 2500; 1000; 316; 100, 31.6 and 10 μg/plate were examined in the Preliminary Concentration Range Finding Test. Based on the results of the preliminary experiment, the examined test concentrations in the Initial Mutation Test, in the Confirmatory Mutation Test and in the Complementary Initial Mutation Test in Salmonella typhimurium TA1537 strain with and without metabolic activation were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

In the Initial Mutation Test, Confirmatory Mutation Tests and the Complementary Initial Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the vehicle control and were within the normal biological variability of the test system.

 

Inhibitory, cytotoxic effects of the test item were not detected in the Preliminary Concentration Range Finding Test and in the main tests in all examined strains with and without metabolic activation.

 

Precipitate/slight precipitate was detected on the plates in the Preliminary Concentration Range Finding Test, in the Initial Mutation Test, Confirmatory Mutation Test in all examined bacterial strains with and without metabolic activation at several concentrations.

 

The mean values of revertant colonies of the negative (vehicle/solvent) control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analysable concentrations were presented in all strains of the main tests, the examined concentration range was considered to be adequate. The study was considered to be valid.

Chromosome aberration test

The key study investigated structural chromosomal aberrations in cultured mammalian cells in accordance with OECD Guidelines for Testing of Chemicals, Section 4, No. 473, “In Vitro Mammalian Chromosome Aberration Test”, 29 July 2016 and Commission Regulation (EU) 2017/735 of 14 February 2017 B.10. "Mutagenicity – In Vitro Mammalian Chromosome Aberration Test".

 

The test material was testedin vitroin a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in Acetone and it was examined up to cytotoxic concentrations and/or solubility limit according to the OECD guideline recommendations. In the performedindependent Chromosome Aberration Assays using duplicate cultures at least 300 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each test item treated, negative (vehicle) and positive control sample.

In Chromosome Aberration Assay 1, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 1000, 333.3, 111.1, 37.04 and 12.35 μg/mL (experiment with and without metabolic activation).

In Assay 1, insolubility (precipitate / minimal amount of precipitate) was detected at the beginning and at the end of the treatment period in the final treatment medium in the 1000-111.1 μg/mL concentration range with and without metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in any samples of this assay. Therefore, concentrations of 333.3, 111.1 and 37.04 μg/mL (a total of three) were chosen for evaluation in the experiment with and without metabolic activation.

 

In Chromosome Aberration Assay 2, a 3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 1000, 333.3, 111.1, 37.04 and 12.35 μg/mL (experiment with and without metabolic activation).

In Assay 2, similarly to the first experiment, insolubility (precipitate / minimal amount of precipitate) was detected at the beginning and at the end of the treatment period in the final treatment medium in the 1000 to 111.1 μg/mL concentration range without metabolic activation and in the 1000 to 37.04 μg/mL concentration range with metabolic activation. There were no large changes in the pH and osmolality. No cytotoxicity was observed in any samples of this assay. Concentrations of 333.3, 111.1 and 37.04 μg/mL (a total of three) were evaluated in the experiment without metabolic activation, and concentrations of 111.1, 37.04 and 12.35 μg/mL (a total of three) were evaluated in the experiment with metabolic activation.

None of the treatment concentrations caused a biologically or statistically significant increase in the number of cells with structural chromosome aberrations in either assay with or without metabolic activation when compared to the appropriate negative (vehicle) control values.

 

Polyploid metaphases (1) were found in some cases in the negative (vehicle) control or test item treated samples in the performedexperiments, but their incidence was not related to treatment with the test item. No endoreduplicated metaphases were detected in the performed experiments.

The negative (vehicle) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.

 

The test item did not induce a significant level of chromosome aberrations in Chinese hamster V79 cells in the performed experiments with and without metabolic activation. Therefore, the test item was not considered clastogenic in this test system.

The experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test item demonstrated no mutagenic activity in the bacterial tester strains used in the study.

Mouse lymphoma assay

The key study followed the procedures indicated by OECD Guidelines for the Testing of Chemicals, Section 4, No. 490, "In Vitro Mammalian Cell Gene Mutation Test using the Thymidine Kinase Gene", 29 July 2016 and Commission Regulation (EC) No. 440/2008 of 30 May 2008, B.17. "Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test" (Official Journal L 142, 31/05/2008).

 

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of test item to cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

 

Acetone was used as vehicle of the test item in this study. The test item was examined up to 1000 μg/mL (the maximum achievable concentration due to the limited solubility of the test item) in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:

(i) Assay 1: 3-hour treatment with metabolic activation: 1000, 333.33, 111.11, 37.04, 12.35 and 4.12 μg/mL

(ii) Assay 1: 3-hour treatment without metabolic activation: 1000, 333.33, 111.11, 37.04, 12.35 and 4.12 μg/mL

(iii) Assay 2: 3-hour treatment with metabolic activation: 1000, 333.33, 111.11, 37.04, 12.35 and 4.12 μg/mL

(iv) Assay 2: 24-hour treatment without metabolic activation: 1000, 333.33, 111.11, 37.04, 12.35 and 4.12 μg/mL

In Assays 1-2, there were no large changes in pH or osmolality after treatment. Insolubility was detected in the final treatment medium at the end of the treatment in the 37.04 - 1000 μg/mL concentration range (treatment with metabolic activation and long treatment without metabolic activation) and 12.35 - 1000 μg/mL concentration range (short treatment without metabolic activation).

 

In Assay 1, following a 3-hour treatment with metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 1000 μg/mL and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase was seen at any examined concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. Therefore, this experiment was considered as being negative.

In Assay 1, following a 3-hour treatment without metabolic activation, no cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 1000 μg/mL and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase in the mutation frequency was observed at any examined concentrations. No significant dose response to the treatment was indicated by the linear trend analysis. This experiment was considered as being negative.

 

In Assay 2, following a 3-hour treatment with metabolic activation, similarly to the first test, no cytotoxicity of the test item was observed. An evaluation was made using data of the highest concentration of 1000 μg/mL and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increase was seen at any evaluated concentrations. No dose-response to the treatment was indicated by the linear trend analysis. This result was considered as confirming the negative effect observed in the first main test in the experiment with metabolic activation.

In Assay 2, following a 24-hour treatment without metabolic activation, no cytotoxicity was observed at any test item treated concentrations. An evaluation was made using data of the highest concentration of 1000 μg/mL and five lower concentrations (a total of six concentrations). No statistically significant or biologically relevant increases in the mutation frequency were seen at any evaluated concentrations. No dose-response

to the treatment was indicated by the linear trend analysis. This experiment confirmed the negative results seen in the first main test in the experiment without metabolic activation.

 

The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were acceptable in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.

 

No mutagenic effect of the test item was observed either in the presence or in the absence of metabolic activation system under the conditions of the Mouse Lymphoma Assay.

In vivo

Negative results were obtained during investigation of in vitro gene mutation in bacteria (Ames test), in vitro cytogenicity in mammalian cells (chromosome aberration study) and in vitro gene mutation in mammalian cells (mouse lymphoma assay). As a result, and in accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 6.0; July 2017), the substance is considered to be non-genotoxic and no further testing is required.

Justification for classification or non-classification

Key in vitro tests demonstrated that the test material was non-mutagenic (Ames test and mouse lymphoma assay) and non-clastogenic (chromosome aberration test). In accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 6.0; July 2017), the substance is not considered to be genotoxic, in vivo testing is not required, and classification in accordance with Regulation (EC) No 1272/2008 does not apply.