Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of test material, either with or without metabolic activation (S9-mix) and an analogue test item was considered to be non-mutagenic (OECD 471, EU Method B.13/14, OPPTS 870.5100 and relevant Japanese guidelines).

 

Mouse lymphoma assay: No mutagenic effect of an analogue test item was observed in the presence or in the absence of metabolic activation(OECD 490 and EU Method B.17).

Chromosome aberration test: An 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).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 June 2016 to 18 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
study report issued later than planned with no impact on results or integrity of the investigation
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
study report issued later than planned with no impact on results or integrity of the investigation
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
yes
Remarks:
study report issued later than planned with no impact on results or integrity of the investigation
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
yes
Remarks:
study report issued later than planned with no impact on results or integrity of the investigation
GLP compliance:
yes (incl. certificate)
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:
S9 fraction prepared from the livers of phenobarbital / β-naphthoflavone induced rats
Test concentrations with justification for top dose:
RANGE-FINDING TEST
- Test item concentrations: 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate.

INITIAL MUTATION TEST AND CONFIRMATORY MUTATION TEST (Salmonella typhimurium TA98, TA100, TA1535 and TA1537)
- Test item concentrations: 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.

INITIAL MUTATION TEST (Escherichia coli WP2 uvrA )
- Test item concentrations: 5000, 1581, 500, 158.1, 50 and 15.81 μg/plate.

CONFIRMATORY MUTATION TEST (Escherichia coli WP2 uvrA )
- Test item concentrations: 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg/plate,

COMPLEMENTARY CONFIRMATORY MUTATION TEST (Salmonella typhimurium TA1535 with metaabolic activation)
- Test item concentration: 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg/plate.
Vehicle / solvent:
N,N-dimethylformamide (DMF)
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylenediamine
Remarks:
4 µg/plate for Salmonella TA98 without metabolic activation
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
2 µg/plate for Salmonella TA100 and TA1535 without metabolic activation
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
50 µg/plate for Salmonella TA1537 without metabolic activation
Positive controls:
yes
Positive control substance:
other: methyl methanesulphonate
Remarks:
2 µL/plate for E coli WP2 uvrA without metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
2 µg/plate for all Salmonella strains with metabolic activation
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
50 µg/plate for E coli WP2 uvrA with metabolic activation
Negative solvent / vehicle controls:
yes
Remarks:
N,N-dimethylformamide
Remarks:
Sigma Aldrich; batch SZBE1830V (purity 100 %; expiry date 16 June 2017) and batch 15K060521 (purity 99.9 %;expiry date 31 October 2020)
Negative solvent / vehicle controls:
yes
Remarks:
distilled water
Remarks:
TEVA Hungary Co (batch 7170914; expiry date 30 September 2017)
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (water ≤ 0.02 %)
Remarks:
Sigma Aldrich batch SZBF3070V (purity 99.99 %; expiry date 18 October 2018) and batch SZBG0550V (purity 99.98 %; expiry date 08 February 2019)
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 examined in a preliminary compatibility test.
- The vehicle N,N-dimethylformamide (DMF) was used to prepare the stock formulation of the test material.
- Test formulations were freshly prepared at the beginning of the experiments in the testing laboratory by diluting the stock formulation using the selected vehicle.
- The respective test concentrations in the main tests are shown in Table 1 (below).
- Analytical determination of the test item concentration, stability and homogeneity was not performed because of the character and the short period of study.

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.

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.
- DMSO (batch SZBF3070V) was used in the preliminary concentration range finding test.
- DMF (batch 15K060521) was used in the complementary confirmatory mutation test.

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 summarized 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.
PROCEDURE FOR GROWING CULTURES
- The frozen bacterial cultures were thawed at room temperature and 200 μL inoculum was 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 6 (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.
- Merck minimal glucose agar plates (batch 138348; expiry date 06 September 2016) was used in the preliminary, batch 138706 (expiry date 27 September 2017) was used in the confirmatory mutation test and batch number 139474 (expiry 13 November 2016) was used in the complementary confirmatory mutation test. Certificates of Analysis were 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.
- Agar solution 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 summarized in Table 6 (attached).

INDUCTION OF LIVER ENZYMES
- Male Wistar rats (385-450 g, animals were 11 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 January 2016.

PREPARATION OF RAT LIVER HOMOGENATE S9 FRACTION
- On Day 4, the rats were euthanized 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 homogenized.
- 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 ºC.
- The date of preparation of S9 fraction for this study was 28 January 2016 (CiToxLAB code: E12297).
- 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 30.8 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.
- 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).
- 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.

TEST PROCEDURE
- The study included a preliminary compatibility test, a preliminary range fnding test (informatory toxicity test), an initial mutation test, a confirmatory mutation test and a complementary confirmatory mutation test.
- At the request of the Sponsor the plate incorporation method was used during the whole study.

CONCENTRATIONS
- Concentrations were selected on the basis of the preliminary compatibility test and preliminary range finding test (informatory toxicity test).

PRELIMINARY COMPATIBILITY TEST
- Solubility of the test item was examined using Distilled water, Dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF).
- The test item was insoluble at 100 mg/mL concentration using Distilled water. Partial dissolution was observed at the same concentration using DMSO as vehicle. However the formulation at the same concentration using DMF was a solution and was suitable for the test. Therefore DMF was selected as vehicle for the test.
- The obtained stock formulation (50 μL) with the solution of top agar (5.4.4.) and phosphate buffer was examined in a test tube without test bacterium suspension. The results of the Solubility Test are summarized in Table 4 (below).

PRELIMINARY CONCENTRATION RANGE-FINDING TEST (INFORMATORY TOXICITY TEST)
- Based on the solubility test, 100 mg/mL stock formulation was prepared in DMF 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 tests, 100 mg/mL stock formulation was prepared from the test item with DMF, which was diluted by serial dilutions in several steps to obtain nine or ten dosing formulations.
- The maximum test concentration was 5000 or 1581 or 500 μg test item/plate in the initial mutation test and confirmatory mutation tests.
- Examined concentrations in the initial mutation test and confirmatory mutation test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains were 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate.
- Examined concentrations in the initial mutation test in the Escherichia coli WP2 uvrA strain were 5000, 1581, 500, 158.1, 50 and 15.81 μg test item/plate.
- Examined concentrations in the confirmatory mutation test in the Escherichia coli WP2 uvrA strain were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate in Escherichia coli WP2 uvrA strain.
- Examined concentrations in the complementary confirmatory mutation test were 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581μg test item/plate for the Salmonella typhimurium TA1535 bacterial strain with metabolic activation.

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 5 (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, confirmatory mutation test and complementary 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.
- Molten top agar was prepared and kept at 45 °C.
- Top agar (2 mL) was aliquoted into individual test tubes (3 tubes per control or concentration level).
- The equivalent number of minimal glucose agar plates were 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 formulation or reference controls (50 µL); overnight culture of test strain (100 µL); phosphate buffer (pH 7.4) or S9 mix (500 µL).
- The solution was mixed and poured on the surface of minimal agar plates.
- For studies with metabolic activation, instead of phosphate buffer, the S9 mix (0.5 mL) was added to each overlay tube.
- The entire test consisted of non-activated and activated test conditions, with the addition of untreated, negative (vehicle/solvent) and positive controls.
- After preparation, the plates were incubated at 37 °C for 48 hours.

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 and 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 Excel software.
- Mutation factor (MF): mean number of revertants on the test item plate / mean number of revertants on the vehicle control plate.
Evaluation criteria:
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.
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRELIMINARY RANGE FINDING TEST (INFORMATORY TOXICITY TEST)
- In the preliminary range finding test, the plate incorporation method was used.
- The preliminary test was performed using Salmonella typhimurium TA98 and Salmonella typhimurium TA100 tester 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.
- In the range finding test the concentrations examined were: 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate.
- Inhibitory, cytotoxic effects of the test item (absent/reduced and/or reduced number of revertant colonies, in some cases pinpoint colonies were also detected) were observed in the preliminary range finding test in Salmonella typhimurium TA98 and TA100 strains without metabolic activation at 5000, 2500, 1000, 316 and 100 μg/plate concentrations and in both strains with metabolic activation at 5000, 2500, 1000 and 316 μg/plate concentrations.
- No precipitate of the test item was detected in the Preliminary Range Finding Test.
- Plates had whitish discoloration at the concentration of 5000 μg/plate in both tested strains with and without metabolic activation.
- The experimental results (revertant colony numbers per plate, mutation factors and standard deviations) are detailed in Table 7 and Appendix 3 (attached).

INITIAL AND CONFIRMATORY MUTATION TESTS
- In the Initial mutation test on Escherichia coli WP2 uvrA bacterial strain with and without metabolic activation no inhibitory or cytotoxic effect of the test item was observed.
- According to the test guidelines the maximum test concentration for soluble non-cytotoxic substances should be 5000 µg test item/plate. Therefore the initial mutation test on Escherichia coli WP2 uvrA bacterial strain with and without metabolic activation were considered to be invalid and was repeated to examine the 5000 µg test item/plate concentration as well. Results of the invalid experiment on this strain are not reported; however, all data were kept and archived in the raw data binder.
- The repeated test with Escherichia coli WP2 uvrA bacterial strain with and without metabolic activation was considered as part of the initial mutation test.
- The confirmatory mutation test on Salmonella typhimurium TA1535 bacterial strain with metabolic activation did not meet the validity criteria; therefore, an additional experiment (complementary confirmatory mutation test) was performed in this strain in an additional experimental period (Experimental Period III) to complete the data. The experimental conditions (except concentrations) were the same as in the confirmatory mutation test. Results of the invalid experiment were not reported; however, all data was kept and archived in the raw data binder.
- In the initial mutation test and confirmatory mutation test none of the observed revertant colony numbers were above the respective biological threshold value. There were no reproducible dose-related trends and no indication of any treatment effect.
- In the initial mutation test, the highest revertant rate was observed in Salmonella typhimurium TA1537 bacterial strain without metabolic activation at the concentration of 5 µg/plate. The mutation factor value was 1.62. However, there was no 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 tests, the highest revertant rate was observed in Escherichia coli WP2 uvrA bacterial strain without metabolic activation at 1.581 µg/plate concentration. The mutation factor value was 1.31. However, the observed mutation factor values were below the biologically relevant threshold limit and the numbers of revertant colonies were within the historical control range.
- Slightly higher numbers of revertant colonies compared to the solvent control were detected in the initial mutation test and in the confirmatory mutation tests sporadically. However, the numbers of revertant colonies were below the biologically relevant threshold value in all cases, so they were considered as reflecting the biological variability of the test.
- Slightly lower revertant counts compared to the solvent control were observed in the initial mutation test and in the confirmatory mutation tests at some non-cytotoxic concentrations. However, the mean numbers of revertant colonies were in the historical control range in all cases.
- Inhibitory, cytotoxic effects of the test item (absent/reduced/slightly reduced background lawn development and/or reduced number of revertant colonies, in some cases pinpoint colonies were also detected) were observed in the initial mutation test in Salmonella typhimurium TA98 and TA1535 strains at 500 and 158.1 μg/plate concentrations with and without metabolic activation, in Salmonella typhimurium TA100 and TA1537 strains at 500, 158.1 and 50 μg/plate concentrations without metabolic activation, in these strains at 500 and 158.1 μg/plate concentrations with metabolic activation.
- Similarly, inhibitory, cytotoxic effects of the test item (absent/reduced/slightly reduced background lawn development and/or reduced number of revertant colonies, in some cases pinpoint colonies were also detected) were observed in the confirmatory mutation test and complementary confirmatory mutation test in Salmonella typhimurium TA98 strain at 500 and 158.1 μg/plate concentrations without metabolic activation, in this strain at 500 μg/plate concentration with metabolic activation, in Salmonella typhimurium TA100 and TA1537 strains at 500, 158.1 and 50 μg/plate concentrations without metabolic activation, in these strains at 500 and 158.1 μg/plate concentrations with metabolic activation, in Salmonella typhimurium TA1535 strain at 5000, 1581, 500 and 158.1 μg/plate concentrations with and without metabolic activation.
- No precipitate was detected on the plates in the main tests in any examined bacterial strain with and without metabolic activation.
- The experimental results (revertant colony numbers per plate, mutation factors, and standard deviations) are summarized in Tables 8-9 and Appendices 4, 5 and 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 (vehicle/solvent) and positive control plates were within the historical control range.
- At least five analysable concentrations were presented in all strains of the main tests.
- The reference mutagens showed a distinct increase of induced revertant colonies.
- The viability of the bacterial cells was checked by a plating experiment in each test.
- The tests were considered to be valid.
Remarks on result:
other: No precipitate was detected on the plates
Conclusions:
Under the experimental conditions applied the test item did not induce gene mutations by basepair changes or frameshifts in the genome of the strains used. The test item demonstrated no mutagenic activity in the applied bacterial tester strains under the test conditions 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 WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavoneinduced rats.

 

The study included a preliminary solubility 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 throughout the study.

 

Based on the results of the solubility test and available information, the test item was formulated in N,N-dimethylformamide (DMF). Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the range finding test. Examined concentrations in the initial mutation test and confirmatory mutation test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains were 500, 158.1,50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate. Examined concentrations in Escherichia coli WP2 uvrA strain in the initial mutation test were 5000, 1581, 500, 158.1, 50 and 15.81 μg test item/plate and in the confirmatory mutation test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate. Examined concentrations in the complementary confirmatory mutation test were 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/platein Salmonella typhimurium TA1535 bacterial strain with metabolic activation.

 

RESULTS

In the initial mutation test and confirmatory mutation tests, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends.

 

Inhibitory, cytotoxic effects of the test item were observed in the initial mutation test and repeatedly in the confirmatory mutation test and complementary confirmatory mutation test for Salmonella typhimurium bacterial strains in the higher concentration range.

 

The mean values of revertant colonies of the 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 tests were considered to be valid.

 

CONCLUSION

Under 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 applied bacterial  tester strains under the test conditions used in the study.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13.
Reason / purpose:
read-across source
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: No precipitate was detected on the plates
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 September 2016 to 06 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: chromosome aberration
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Rat Liver Homogenate S9 Fraction
Test concentrations with justification for top dose:
- Experiment 1 (without metabolic activation): 25, 20, 15, 10, 5.0, 2.5 and 1.25 μg/mL
- Experiment 1 (with metabolic activation): 30, 20, 15, 10, 5.0, 2.5 and 1.25 μg/mL
- Experiment 2 (without metabolic activation): 20, 15, 10, 5, 2.5, 1.25 and 0.625 μg/mL
- Experiment 2 (with metabolic activation): 25, 20, 15, 10, 5, 2.5 and 1.25 μg/mL
Negative solvent / vehicle controls:
yes
Positive control substance:
other: Dimethyl sulfoxide
Remarks:
Sigma Aldrich; Lot SZBG0550V (preliminary study) / SZBG1310V (main test); Expiry dates 08 February 2019 / 25 April 2019; Stored at room temperature under nitrogen)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
ethylmethanesulphonate
Remarks:
EMS (Sigma Aldrich; Lot BCBN1209V; Expiry date 31 May 2017; Stored at room temperature under nitrogen
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
cyclophosphamide
Remarks:
Sigma Aldrich; Lot MKBX1822V; Expiry date 31 December 2018; Stored refrigerated (2 to 8 °C)
Details on test system and experimental 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.

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 (200 mg/mL in the preliminary experiment, 10
mg/mL in the main tests) were prepared 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 formulation was stirred until homogeneity was reached.
- The stock solution was filtered sterile using a 0.22 μm syringe filter (Supplier: Millipore, Lot R5HA09546, Expiry date: June 2018).
- From the stock formulation several dilutions were prepared using the selected vehicle (solvent) to prepare dosing formulations.
- The dosing formulations (clear solutions) 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/solvent) control samples were run concurrently with treatment groups.
- Based on the result of a short solubility test, the test tem was insoluble
in Distilled water at 200 mg/mL concentration, but it was soluble in Dimethyl sulfoxide
(DMSO) at the same concentration. Therefore, DMSO was selected as vehicle
(solvent) of the study.
- The vehicle (solvent) was compatible with the survival of the cells and the S9 activity.

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: R5HA09546, Expiry date: June 2018).

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 (322-391 g, animals were 9 weeks old at the initiation of E12075; 345 to 441 g, animals were 9 weeks old at the initiation of E12440) 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 were 30 March 2015 (E12075) and 25 July 2016 (E12440).

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 ± 10 °C. The dates of preparation of S9 fractions for this study were 02 April 2015 (E12075) and 28 July 2016 (E12440) (note: CiToxLAB lot of E12075 was used in the preliminary experiment; CiToxLAB lot of E12440 was used in the main tests).
- 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 fractions used in the study was determined to be 28.0 g/L (E12075) and 29.2 g/L (E12440). 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 3 hours in the presence of S9-mix and for 20 hours in the absence of S9-mix with a 28-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 3 (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 28-hour after the beginning of the treatment.

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) or 28 hours (approximately 2 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 8 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 was conducted under the control of the Principal Investigator. When the metaphase analysis was completed for each test, the slide codes were broken and the number of metaphases with aberrations (excluding gaps) and the types of aberrations for each culture were presented in tables.
- 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.
- Note: The examination of slides from a culture was halted when 25 or more metaphases with aberrations (excluding gaps) have been recorded for that culture.
- 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.
- 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.
- The metaphase analysis was conducted in compliance with Good Laboratory Practice as required by the United Kingdom GLP Compliance Regulations 1999 (SI 1999 No. 3106, as amended 2004, SI No. 0994) and which are in compliance with the OECD Principles of Good Laboratory Practice (as revised in 1997). These Principles are in conformity with other international GLP regulations.
- The Quality Assurance Unit at the Test Site performed study and/or process-based inspections according to its own Standard Operating Procedures. Inspection results were reported promptly in writing to the Principal Investigator who subsequently informed the Study Director. The inspection results were also communicated to the Lead QA (CiToxLAB Hungary Ltd.) and Test Facility Management. The Test Site QA provided a QA statement for inclusion in the study report.
- The Principal Investigator received the signed Study Plan and amendment as PDF-files by e-mail, and distributed them as required at the Test Site. All study related communication between the Study Director, Principal Investigator and Sponsor or Sponsor’s representative were documented and archived with the raw data.
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 6 (attached).
Statistics:
STATISTICAL ANALYSIS
- 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 Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
VEHICLE AND CONCENTRATION SELECTION
- Based on the result of a short solubility test, the test tem was insoluble in Distilled water at 200 mg/mL concentration, but it was soluble in Dimethyl sulfoxide (DMSO) at the same concentration. Therefore, DMSO was selected as vehicle (solvent) of the study. The highest examined concentration in the preliminary test was 2000 μg/mL.
- Two Concentration Selection Cytotoxicity Assays (Assay A: 3-hour treatment with and without metabolic activation, 20-hour harvesting time; and Assay B: 3-hour treatment with metabolic activation or 20-hour treatment without metabolic activation, 28-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 2000 and 3.906 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, 4 and 5 of Appendix 3 (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. (up to the cytotoxicity limit).

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 25, 20, 15, 10, 5, 2.5 and 1.25 μg/mL (experiment without metabolic activation); and 30, 20, 15, 10, 5, 2.5 and 1.25 μg/mL (experiment with metabolic activation).
- In Assay 1, no insolubility was detected. There were no large changes in the pH and osmolality. Strong cytotoxicity was observed in the experiment with and without metabolic activation (RICC value of the highest evaluated concentration was 51% in the experiment with metabolic activation and 33 % in the experiment without metabolic activation) as detailed in Tables 6 and 7 of Appendix 4. Therefore, concentrations of 20, 10 and 5 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 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 20, 15, 10, 5, 2.5, 1.25 and 0.625 μg/mL (experiment without metabolic activation); and 25, 20, 15, 10, 5, 2.5 and 1.25 μg/mL (experiment with metabolic activation).
- In Assay 2, similarly to the first experiment, no insolubility was detected. There were no large changes in the pH and osmolality. Strong cytotoxicity was observed in the experiment with and without metabolic activation (RICC value of the highest evaluated concentration was 25 % in the experiment with metabolic activation and 36 % in the experiment without metabolic activation) as detailed in Tables 8 and 9 of Appendix 4. Concentrations of 15, 10, and 5 μg/mL (a total of three) were evaluated in the experiment without metabolic activation, and concentrations of 25, 20, 15 and 10 μg/mL (a total of four) were evaluated in the experiment with metabolic activation.
- None of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations in the experiment with or without metabolic activation when compared with the appropriate negative (vehicle) control and/or untreated control values (see Tables 10-13 of Appendix 5, attached). Based on these facts, the test item was considered to have given a negative response.
- Polyploid metaphases were found in some cases in the negative (vehicle) control, positive control or test item treated samples in the performed experiments. One endoreduplicated metaphases were detected in three test item treated samples in Assay 1, while no endoreduplicated metaphases were observed in Assay 2 in any samples.

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 in the higher concentration range; while strong cytotoxicity was detected in the 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.
- Note: Regarding cytotoxicity, the recent OECD guideline requires that the highest evaluated concentration should have cytotoxicity in the 50 to 60 % range. Although a closely spaced concentration
range was used in the performed experiments, due to the sharp cytotoxicity profile of the test item mostly higher values were achieved: in Assay 1 the highest evaluated concentrations showed cytotoxicity of 49 % and 67 % (with and without metabolic activation respectively), while in Assay 2 the highest evaluated concentrations showed cytotoxicity of 75% and 64% (with and without metabolic activation), the following next concentrations would have been a cytotoxicity well below the limit value. However, as the requested cytotoxic range was covered in those cases, these values (including the marginal difference in Assay 1 with metabolic activation) were considered as acceptable and did not adversely affect the results or interpretation of the study.
- At least three test item concentrations were evaluated in each experiment. Concentrations were separated by a factor of 2, or were more closely spaced in the expected cytotoxic concentration range.Historical control data are presented in Appendix 6 (attached). The spontaneous aberration frequencies of the negative (vehicle/solvent) control and/or untreated control samples in the performed experiments were considered to be acceptable based on the historical control range of the testing laboratory. Note: In Assay 1 with metabolic activation, aberration level of the negative (vehicle / solvent) control was outside the historical control range of that harvesting time-point, (due to one outlier replicate with higher than usual aberration frequency), thus the untreated control was additionally analysed. The number of aberrant cells in the negative (vehicle/solvent) control was marginally statistically significantly higher than the untreated control (p = 0.048), so the untreated control value was used for comparison with test item treated samples in this test instead of the negative (vehicle/solvent) control. This fact was considered not to adversely affect the results or integrity of the study since we have historical data showing that solvent and untreated controls are not significantly different.
- 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 (Tables 10-13 of Appendix 5, attached) demonstrating the sensitivity of the test system in each assay except of Assay 1 without metabolic activation. However, as according to the recent OECD No. 473 guideline, the appropriate results of a single positive control substance requiring metabolic activation demonstrates both the activity of the metabolic activation system and the responsiveness of the test system, this fact was considered not to adversely affect the validity of the study.
- 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 VitroMammalian 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 Dimethyl sulfoxide (DMSO) 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, untreated control, negative (vehicle/solvent) 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 25, 20, 15, 10, 5.0, 2.5 and 1.25 μg/mL (experiment without metabolic activation); and 30, 20, 15, 10, 5.0, 2.5 and 1.25 μg/mL (experiment with 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 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test item were 20, 15, 10, 5, 2.5, 1.25 and 0.625 μg/mL (experiment without metabolic activation); and 25, 20, 15, 10, 5, 2.5 and 1.25 μg/mL (experiment with metabolic activation).

 

In Assay 1, no insolubility was detected. There were no large changes in the pH and osmolality. Strong cytotoxicity was observed in the experiment with and without metabolic activation (RICC – Relative Increase in Cell Counts - value of the highest evaluated concentration was 51% in the experiment with metabolic activation and 33 % in the experiment without metabolic activation). Therefore, concentrations of 20, 10 and 5 μg/mL (a total of three) were chosen for evaluation in the experiment with and without metabolic activation.

 

In Assay 2, similarly to the first experiment, no insolubility was detected. There were no large changes in the pH and osmolality. Strong cytotoxicity was observed in the experiment with and without metabolic activation (RICC value of the highest evaluated concentration was 25 % in the experiment with metabolic activation and 36 % in the experiment without metabolic activation). Concentrations of 15, 10, and 5 μg/mL (a total of three) were evaluated in the experiment without metabolic activation, and concentrations of 25, 20, 15 and 10 μg/mL (a total of four) were evaluated in the experiment with metabolic activation.

 

RESULTS

None of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations in the experiment with or without metabolic activation when compared with the appropriate negative (vehicle/solvent) control and/or untreated control values. Based on these facts, the test item was considered to have given a negative response.

 

Polyploid metaphases were found in some cases in the negative (vehicle/solvent) control, positive control or test item treated samples in the performed experiments. One endoreduplicated metaphases were detected in three test item treated samples in Assay 1, while no endoreduplicated metaphases were observed in Assay 2 in any samples.

 

The negative (vehicle/solvent) control and/or untreated control data were within the acceptable range for the spontaneous aberration frequency, the positive control substance with and/or without metabolic activation caused a statistically significant increase in the number of structural aberrations excluding gaps demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; at least 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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13.
Reason / purpose:
read-across source
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 August 2016 to 22 September 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
other: mouse lymphoma assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital / ß-Naphtha flavone induced S9 mix
Test concentrations with justification for top dose:
- Assay 1, 3-hour treatment with metabolic activation: 70, 60, 50, 40, 30, 20, 10, 5 and 2.5 μg/mL,
- Assay 1, 3-hour treatment without metabolic activation: 40, 35, 30, 25, 20, 15, 10, 5 and 2.5 μg/mL μg/mL,
- Assay 2, 3-hour treatment with metabolic activation: 70, 60, 50, 40, 30, 20, 10, 5 and 2.5 μg/mL,
- Assay 2, 24-hour treatment without metabolic activation: 35, 30, 25, 20, 15, 10, 5, 2.5 and 1.25 μg/mL μg/mL.
Vehicle / solvent:
Dimethyl sulfoxide
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl sulfoxide (10 μL/mL for Assays 1 and 2)
Remarks:
DMSO (Sigma Aldrich; Lot SZBG0550V; Expiry 08 February 2019; Room temperature storage under nitrogen)
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Sigma Aldrich; Lot WXBB6231V; Expiry 30 June 2016; Storage conditions -23 ± 8 °C. Stock solution was prepared using the pure chemical (powder) on 06 June 2016 and kept frozen (Expiry date: 06 December 2016).
Positive controls:
yes
Remarks:
with metabolic activation
Positive control substance:
cyclophosphamide
Remarks:
Sigma Aldrich; Lot MKBX1822V; Expiry date 31 December 2018; Storage conditions 2 to 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 available information, the test item was insoluble in distilled water at 200 mg/mL concentration, but was soluble in dimethyl sulfoxide (DMSO) at the same concentration. Therefore, DMSO was selected for vehicle (solvent) 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 solutions (200 mg/mL, clear solution) were prepared as follows. The necessary amount of test item was weighed into a calibrated volumetric flask; approximately 80% of the required volume of the vehicle (solvent) was added and vigorously stirred to obtain a homogenous formulation. It was filled up to the final volume with the vehicle to form a stock solution. From the stock solution several dilutions were prepared using the selected vehicle (solvent) to prepare dosing formulations. In each case, the stock solution and the solvent was filtered sterile using a 0.22 μm syringe filter (Supplier: Millipore, Lot No.: R5HA09546, Expiry date: June 2018) before the preparation of the dosing formulations. The stock solutions and all the dosing formulations were prepared immediately before the treatment of the cells in the testing laboratory (in a sterile hood). 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.

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 °C ± 10 °C. Aliquots were removed from the ultrafreezer immediately 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.
- Cyclophosphamide, a genotoxic agent that requires metabolic transformation by microsomal enzymes, was dissolved in DMSO immediately 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.

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

TEST SYSTEM
- Cell line: L5178Y TK+/- 3.7.2 C mouse lymphoma
- Product number: CRL-9518
- Lot number: 1661603
- Date of receipt: 22 January 2004
- Date of working lot: 21 January 2016
- 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).

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 (385-450 g, animals were 11 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 January 2016.
- Preparation of Rat Liver Homogenate S9 Fraction: On Day 4, the rats were euthanized 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 9000g 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 30.8 g/L. The date of preparation of S9 fraction for this study was 28 January 2016 (CiToxLAB code: E12297). 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, 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°C ± 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.

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 2 x 10E05 cells/mL and transferred to flasks for further growth.
- On completion of the expression period, at least seven 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 °C ± 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 2x10E03 cells per well.
- Microplates were incubated at 37 °C ± 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.
- Caution was exercised with positive results obtained at levels of cytotoxicity lower than 10% (as measured by RTG).
- 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
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
PRELIMINARY 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 was 2000 μg/mL (the recommended maximum concentration). Tabulated results of the preliminary experiment are given in Appendix 4 (attached).
- Insolubility and cytotoxicity was detected in the preliminary experiments. Therefore, concentrations up to the cytotoxicity limit were selected for the main experiments according to the instructions of the relevant OECD guideline. At least seven 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 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 70, 60, 50, 40, 30, 20, 10, 5, and 2.5 μg/mL (experiment with metabolic activation); and 40, 35, 30, 25, 20, 15, 10, 5 and 2.5 μg/mL (experiment without metabolic activation).
- 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 insolubility or large changes in pH or osmolality were detected in the final treatment medium at the end of the treatment.
- In the presence of S9-mix (3-hour treatment), excessive cytotoxicity of the test item was observed: cells of the 70 and 60 μg/mL concentrations died during the treatment or in the expression period (survival results are shown in Table 1 of Appendix 5, attached). An evaluation was made using data of the first surviving concentration of 50 μg/mL (relative total growth: 15%) and six lower concentrations (a total of seven concentrations). Statistically significant increase was seen at 50 μg/mL concentration, however, the increase was biologically not relevant (the difference between the observed value and the negative (vehicle) control relevant data was smaller than the Global Evaluation Factor). 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), marked cytotoxicity of the test item was observed at 40 μg/mL concentration (survival results are detailed in Table 2 of Appendix 5, attached). The relative total growth of this sample was 1%, therefore it was excluded from the evaluation. An evaluation was made using the eight remaining concentrations (the relative total growth of the highest evaluated concentration of 35 μg/mL was 8%). No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis (for details see
Table 10 of Appendix 7, attached). This experiment was 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 70, 60, 50, 40, 30, 20, 10, 5, and 2.5 μg/mL (experiment with metabolic activation); and 35, 30, 25, 20, 15, 10, 5, 2.5 and 1.25 μg/mL μg/mL (experiment without metabolic activation).
- 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 insolubility or large changes in pH or osmolality were detected in the final treatment medium at the end of the treatment.
- In the presence of S9-mix (3-hour treatment), similarly to the first test, excessive cytotoxicity of the test item was observed at 70 and 60 μg/mL concentrations, cells of these samples died during the treatment or in the expression period (survival results are shown in Table 3 of Appendix 5, attached). An evaluation was made using data of the first surviving concentration of 50 μg/mL (relative total growth: 2%) and six lower concentrations (a total of seven concentrations). No statistically significant or biologically relevant increase was seen at any evaluated concentration. 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 absence of S9-mix (24-hour treatment), excessive cytotoxicity was observed at 35 and 30 μg/mL concentrations. Cells of these samples died during the treatment (survival results are shown in Table 4 of Appendix 5, attached). An evaluation was made using data using data of the first surviving concentration of 25 μg/mL (relative total growth: 16%) and six lower concentrations (a total of seven concentrations). No biologically relevant or statistically significant increase in the mutation frequency was observed at any evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis (details are shown in Table 12 of Appendix 7). This experiment confirmed the negative result seen in the first main test.
- Some 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 at the end of the expression period (PEviability) were within the acceptable range (65-120%) in all assays (Tables 5-8 of Appendix 6, attached) except of the experiment without metabolic activation in Assay 2 where marginally lower value (64%) was detected for the negative (vehicle) control. However, the plating efficiency of untreated control sample was within the range, thus the observed values were considered to be acceptable.
- The number of test concentrations evaluated was at least seven 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 cytotoxicity limit were examined in the study. In case of cytotoxicity, the defined acceptance criterion regarding cytotoxicity produced by the highest evaluated concentration (approximately 80-90 % toxicity, i.e. approximately 10-20 % relative survival or relative total growth) was considered to be fulfilled. Lower test concentrations were usually spaced by a factor of two, but more closely
spaced concentrations were used in the expected cytotoxic range. Note: In Assay 2, in case of the experiment with metabolic activation, the relative total growth value of the highest surviving concentration of 50 μg/mL was 2%. Although this value showed stronger cytotoxicity than the recommended upper limit, it was selected for evaluation to properly cover the cytotoxic concentration range (the relative total growth value of the next – closely spaced – concentration of 40 μg/mL was 38%). This fact was considered not to adversely affect the results of the study.
- Suspension growth value of the untreated and negative (vehicle) control were slightly lower or lower in some cases than the recommended value (Appendix 17), 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 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 VitroMammalian Cell Gene Mutation Test using the Thymidine Kinase Gene", 28 July 2016 and Commission Regulation (EC) No. 440/2008 of 30 May 2008, B.17. "Mutagenicity -In VitroMammalian 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). Dimethyl sulfoxide was used as vehicle of the test item in this study. The test item was examined up to 2000 μg/mL (the recommended maximum concentration) in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:

- Assay 1, 3-hour treatment with metabolic activation: 70, 60, 50, 40, 30, 20, 10, 5 and 2.5 μg/mL,

- Assay 1, 3-hour treatment without metabolic activation: 40, 35, 30, 25, 20, 15, 10, 5 and 2.5 μg/mL

- Assay 2, 3-hour treatment with metabolic activation: 70, 60, 50, 40, 30, 20, 10, 5 and 2.5 μg/mL

- Assay 2, 24-hour treatment without metabolic activation: 35, 30, 25, 20, 15, 10, 5, 2.5 and 1.25 μg/mL

 

RESULTS

In Assays 1-2, there were no large changes in pH or osmolality after treatment. No insolubility was detected in the final treatment medium at the end of the treatment.

 

In Assay 1, following a 3-hour treatment with metabolic activation, excessive cytotoxicity of the test item was observed: cells of the 70 and 60 μg/mL concentrations died during the treatment or in the expression period. An evaluation was made using data of the first surviving concentration of 50 μg/mL (relative total growth: 15%) and six lower concentrations (a total of seven concentrations). A statistically significant increase was seen at 50 μg/mL concentration, however, the increase was biologically not significant (the difference between the observed value and the negative (vehicle)

control relevant data was smaller than the Global Evaluation Factor). 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, marked cytotoxicity of the test item was observed at 40 μg/mL concentration. The relative total growth of this sample was 1%, therefore it was excluded from the evaluation. An evaluation was made using the eight remaining concentrations (the relative total growth of the highest evaluated concentration of 35 μg/mL was 8%). No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.

 

In Assay 2, following a 3-hour treatment with metabolic activation, similarly to the first test, excessive cytotoxicity of the test item was observed at 70 and 60 μg/mL concentrations, cells of these samples died during the treatment or in the expression period. An evaluation was made using data of the first surviving concentration of 50 μg/mL (relative total growth: 2%) and six lower concentrations (a total of seven concentrations). No statistically significant or biologically relevant increase was seen at any evaluated concentration. 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 Assay 2, following a 24-hour treatment without metabolic activation, excessive cytotoxicity was observed at 35 and 30 μg/mL concentrations. Cells of these samples died during the treatment. An evaluation was made using data using data of the first surviving concentration of 25 μg/mL (relative total growth: 16%) and six lower concentrations (a total of seven concentrations). No biologically relevant or statistically significant increase in the mutation frequency was observed at any evaluated

concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment confirmed the negative result seen in the first main test.

 

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 in the presence or in the absence of metabolic activation system under the conditions of the Mouse Lymphoma Assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13.
Reason / purpose:
read-across source
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
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

An analogue 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 WP2 uvrA) 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 solubility 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 throughout the study.

 

Based on the results of the solubility test and available information, the test item was formulated in N,N-dimethylformamide (DMF). Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the range finding test. Examined concentrations in the initial mutation test and confirmatory mutation test in Salmonella typhimurium TA98, TA100, TA1535 and TA1537 strains were 500, 158.1,50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate. Examined concentrations in Escherichia coli WP2 uvrA strain in the initial mutation test were 5000, 1581, 500, 158.1, 50 and 15.81 μg test item/plate and in the confirmatory mutation test were 5000, 1581, 500, 158.1, 50, 15.81, 5 and 1.581 μg test item/plate. Examined concentrations in the complementary confirmatory mutation test were 5000, 1581, 500, 158.1, 50, 15.81, 5, 1.581, 0.5 and 0.1581 μg test item/plate in Salmonella typhimurium TA1535 bacterial strain with metabolic activation.

 

In the initial mutation test and confirmatory mutation tests, none of the observed revertant colony numbers were above the respective biological threshold value. There were no consistent dose-related trends.

 

Inhibitory, cytotoxic effects of the test item were observed in the initial mutation test and repeatedly in the confirmatory mutation test and complementary confirmatory mutation test for Salmonella typhimurium bacterial strains in the higher concentration range.

The mean values of revertant colonies of the 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 tests were considered to be valid.

 

Under the experimental conditions applied the analogue 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 applied bacterial tester strains under the test conditions 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", 28 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 an analogue 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). Dimethyl sulfoxide was used as vehicle of the test item in this study. The test item was examined up to 2000 μg/mL (the recommended maximum concentration) in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:

- Assay 1, 3-hour treatment with metabolic activation: 70, 60, 50, 40, 30, 20, 10, 5 and 2.5 μg/mL,

- Assay 1, 3-hour treatment without metabolic activation: 40, 35, 30, 25, 20, 15, 10, 5 and 2.5 μg/mL

- Assay 2, 3-hour treatment with metabolic activation: 70, 60, 50, 40, 30, 20, 10, 5 and 2.5 μg/mL

- Assay 2, 24-hour treatment without metabolic activation: 35, 30, 25, 20, 15, 10, 5, 2.5 and 1.25 μg/mL

 

In Assays 1-2, there were no large changes in pH or osmolality after treatment. No insolubility was detected in the final treatment medium at the end of the treatment.

 

In Assay 1, following a 3-hour treatment with metabolic activation, excessive cytotoxicity of the test item was observed: cells of the 70 and 60 μg/mL concentrations died during the treatment or in the expression period. An evaluation was made using data of the first surviving concentration of 50 μg/mL (relative total growth: 15%) and six lower concentrations (a total of seven concentrations). A statistically significant increase was seen at 50 μg/mL concentration, however, the increase was biologically not significant (the difference between the observed value and the negative (vehicle) control relevant data was smaller than the Global Evaluation Factor). 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, marked cytotoxicity of the test item was observed at 40 μg/mL concentration. The relative total growth of this sample was 1%, therefore it was excluded from the evaluation. An evaluation was made using the eight remaining concentrations (the relative total growth of the highest evaluated concentration of 35 μg/mL was 8%). No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.

 

In Assay 2, following a 3-hour treatment with metabolic activation, similarly to the first test, excessive cytotoxicity of the test item was observed at 70 and 60 μg/mL concentrations, cells of these samples died during the treatment or in the expression period. An evaluation was made using data of the first surviving concentration of 50 μg/mL (relative total growth: 2%) and six lower concentrations (a total of seven concentrations). No statistically significant or biologically relevant increase was seen at any evaluated concentration. 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 Assay 2, following a 24-hour treatment without metabolic activation, excessive cytotoxicity was observed at 35 and 30 μg/mL concentrations. Cells of these samples died during the treatment. An evaluation was made using data using data of the first surviving concentration of 25 μg/mL (relative total growth: 16%) and six lower concentrations (a total of seven concentrations). No biologically relevant or statistically significant increase in the mutation frequency was observed at any evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis. This experiment confirmed the negative result seen in the first main test.

 

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 analogue test item was observed in the presence or in the absence of metabolic activation under the conditions of the Mouse Lymphoma Assay.

 

Chromosome aberration test

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 VitroMammalian Chromosome Aberration Test".

An analogue test material was investigated in vitro in a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in Dimethyl sulfoxide (DMSO) 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, untreated control, negative (vehicle/solvent) 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 25, 20, 15, 10, 5.0, 2.5 and 1.25 μg/mL (experiment without metabolic activation); and 30, 20, 15, 10, 5.0, 2.5 and 1.25 μg/mL (experiment with 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 28 hours after the beginning of the treatment

in both cases. The examined concentrations of the test item were 20, 15, 10, 5, 2.5, 1.25 and 0.625 μg/mL (experiment without metabolic activation); and 25, 20, 15, 10, 5, 2.5 and 1.25 μg/mL (experiment with metabolic activation).

 

In Assay 1, no insolubility was detected. There were no large changes in the pH and osmolality. Strong cytotoxicity was observed in the experiment with and without metabolic activation (RICC – Relative Increase in Cell Counts - value of the highest evaluated concentration was 51% in the experiment with metabolic activation and 33 % in the experiment without metabolic activation). Therefore, concentrations of 20, 10 and 5 μg/mL (a total of three) were chosen for evaluation in the experiment with and without metabolic activation.

 

In Assay 2, similarly to the first experiment, no insolubility was detected. There were no large changes in the pH and osmolality. Strong cytotoxicity was observed in the experiment with and without metabolic activation (RICC value of the highest evaluated concentration was 25 % in the experiment with metabolic activation and 36 % in the experiment without metabolic activation). Concentrations of 15, 10, and 5 μg/mL (a total of three) were evaluated in the experiment without metabolic activation, and concentrations of 25, 20, 15 and 10 μg/mL (a total of four) were evaluated in the experiment with metabolic activation.

 

None of the treatment doses caused a significant increase in the number of cells with structural chromosome aberrations in the experiment with or without metabolic activation when compared with the appropriate negative (vehicle/solvent) control and/or untreated control values. Based on these facts, the test item was considered to have given a negative response.

Polyploid metaphases were found in some cases in the negative (vehicle/solvent) control, positive control or test item treated samples in the performed experiments. One endoreduplicated metaphases were detected in three test item treated samples in Assay 1, while no endoreduplicated metaphases were observed in Assay 2 in any samples.

 

The negative (vehicle/solvent) control and/or untreated control data

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

 

The analogue 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.

In vivo

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

Justification for classification or non-classification

Key in vitro tests demonstrated that an analogue 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.