Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay: negative
HPRT gene mutation assay: negative

Micronucleus test in human lymphocytes: negative

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:
2016-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(21 Jul 1997)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
The test item was homogeneous by visual inspection.
Storage conditions: Room temperature
Batch No.: VFH-2016-08
Target gene:
his+ / trp+
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver S9 microsomal fraction
Test concentrations with justification for top dose:
1st Experiment (Standard plate test with and without S9 mix): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
2nd Experiment (Preincubation test with and without S9 mix): 0; 33; 100; 333; 1000; 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

Standard plate test:
- Exposure duration: 48 – 72 hours

Preincubation test:
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer


OTHER:
Titer determination: The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

Positive controls:

With S9 mix: 2-aminoanthracene (2-AA), 2.5 μg/plate, dissolved in DMSO / TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, dissolved in DMSO / Escherichia coli WP2 uvrA
Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 5 μg/plate, dissolved in DMSO / TA 1535, TA 100; 4-nitro-o-phenylenediamine (NOPD), 10 μg/plate, dissolved in DMSO / TA 98; 9-aminoacridine (AAC), 100 μg/plate, dissolved in DMSO / TA 1537; 4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141), 5 μg/plate, dissolved in DMSO / E. coli WP2 uvrA
Evaluation criteria:
Acceptance criteria:
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.

Assessment criteria:
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.
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
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:A weak bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was occasionally observed in the standard plate and preincubation test from about 2500 μg/plate onward.

1st experiment, standard plate incorporation assay without metabolic activation
Strain test group dose (mg/plate) mean revertants per plate standard deviation factor individual colony count
TA 1535 DMSO - 11.3 4.0 - 7, 12, 15
Test item 33 11.7 2.5 1.0 14, 12, 9
100 6.7 3.1 0.6 6, 4, 10
333 10.7 4.0 0.9 10, 15, 7
1000 11.0 3.6 1.0 7, 14, 12
2500 10.7 3.5 0.9 14, 7, 11
5000 10.0 4.6 0.9 5, 14, 11
  MNNG 5.0 5591.7 410.3 493.4 6037, 5509, 5229
TA 100 DMSO - 112.0 14.9 - 118, 95, 123
Test item 33 103.7 9.3 0.9 96, 101, 114
100 109.7 6.5 1.0 103, 110, 116
333 104.0 11.0 0.9 104, 93, 115
1000 111.7 9.1 1.0 122, 105, 108
2500 109.3 5.0 1.0 114, 104, 110
5000 96.7 4.2 0.9 100, 92, 98
  MNNG 5.0 3747.3 403.7 33.5 4168, 3363, 3711
TA 1537 DMSO - 11.0 2.0 - 11, 9, 13
Test item 33 11.3 4.0 1.0 15, 7, 12
100 12.0 3.6 1.1 15, 8, 13
333 12.0 0.0 1.1 12, 12, 12
1000 8.3 3.5 0.8 8, 5, 12
2500 8.7 3.5 0.8 12, 5, 9
5000 11.7 2.5 1.1 9, 12, 14
  AAC 100 949.0 104.8 86.3 885, 1070, 892
TA 98 DMSO - 20.3 5.8 - 17, 27, 17
Test item 33 17.7 6.5 0.9 11, 18, 24
100 18.0 2.0 0.9 18, 16, 20
333 17.0 6.2 0.8 24, 12, 15
1000 17.0 1.7 0.8 16, 16, 19
2500 18.0 6.0 0.9 12, 24, 18
5000 19.0 7.2 0.9 27, 13, 17
  NOPD 10 591.3 9.1 29.1 595, 581, 598
E. coli DMSO - 20.7 2.5 - 21, 18, 23
Test item 33 21.7 3.5 1.0 25, 22, 18
100 26.7 3.2 1.3 28, 29, 23
333 25.0 3.5 1.2 23, 29, 23
1000 21.3 2.5 1.0 24, 21, 19
2500 16.0 2.0 0.8 18, 16, 14
5000 17.3 4.0 0.8 13, 21, 18
  4-NQO 5 1150.0 49.7 55.6 1202, 1145, 1103

1st experiment, standard plate incorporation assay with metabolic activation
Strain test group dose (mg/plate) mean revertants per plate standard deviation factor individual colony count
TA 1535 DMSO - 13.7 4.2 - 9, 17, 15
Test item 33 10.0 2.6 0.7 12, 11, 7
100 10.3 4.9 0.8 7, 8, 16
333 12.0 5.0 0.9 7, 17, 12
1000 9.3 3.1 0.7 6, 12, 10
2500 12.0 6.2 0.9 7, 10, 19
5000 10.3 4.0 0.8 8, 15, 8
  2-AA 2.5 271.3 11.2 19.9 274, 281, 259
TA 100 DMSO - 109.0 10.4 - 97, 115, 115
Test item 33 105.3 6.5 1.0 105, 99, 112
100 112.0 8.7 1.0 102, 117, 117
333 103.7 10.7 1.0 113, 92, 106
1000 94.7 9.7 0.9 103, 97, 84
2500 88.0 4.0 0.8 84, 88, 92
5000 101.0 18.1 0.9 103, 82, 118
  2-AA 2.5 2962.3 124.5 27.2 3086, 2964, 2837
TA 1537 DMSO - 9.7 2.1 - 8, 12, 9
Test item 33 12.3 2.5 1.3 12, 10, 15
100 11.0 1.0 1.1 12, 10, 11
333 12.7 4.5 1.3 13, 8, 17
1000 9.0 2.0 0.9 9, 11, 7
2500 6.3 2.3 0.7 5, 5, 9
5000 4.7 3.1 0.5 4, 8, 2
  2-AA 2.5 219.0 23.6 22.7 229, 236, 192
TA 98 DMSO - 24.0 4.4 - 26, 27, 19
Test item 33 23.7 6.7 1.0 31, 18, 22
100 21.7 4.2 0.9 23, 25, 17
333 22.0 11.3 0.9 35, 16, 15
1000 25.3 12.3 1.1 22, 15, 39
2500 19.0 4.6 0.8 14, 20, 23
5000 23.7 3.1 1.0 27, 21, 23
  2-AA 2.5 2681.7 189.9 111.7 2590, 2555, 2900
E. coli DMSO - 34.3 4.9 - 32, 31, 40
Test item 33 33.0 13.9 1.0 24, 26, 49
100 22.3 1.2 0.7 23, 23, 21
333 25.7 1.5 0.7 26, 27, 24
1000 24.0 7.2 0.7 30, 16, 26
2500 18.3 3.5 0.5 22, 15, 18
5000 16.3 7.8 0.5 10, 25, 14
  2-AA 60 90.3 13.3 2.6 75, 99, 97

2st experiment, preincorporation assay without metabolic activation
Strain test group dose (mg/plate) mean revertants per plate standard deviation factor individual colony count
TA 1535 DMSO - 10.3 3.1 - 11, 7, 13
Test item 33 11.7 1.5 1.1 12, 10, 13
100 11.0 4.6 1.1 7, 10, 16
333 7.7 0.6 0.7 8, 7, 8
1000 9.7 0.6 0.9 10, 9, 10
2500 11.3 3.5 1.1 15, 8, 11
5000 6.0 5.3 0.6 2, 12, 4
  MNNG 5.0 2405.0 253.9 232.7 2673, 2168, 2374
TA 100 DMSO - 99.3 16.2 - 89, 91, 118
Test item 33 103.7 10.0 1.0 100, 96, 115
100 89.7 8.4 0.9 80, 94, 95
333 92.3 6.4 0.9 97, 95, 85
1000 79.3 8.1 0.8 84, 84, 70
2500 94.0 6.6 0.9 101, 93, 88
5000 71.0 7.5 0.7 70, 79, 64
  MNNG 5.0 2405.3 260.1 24.2 2414, 2141, 2661
TA 1537 DMSO - 6.7 0.6 - 7, 7, 6
Test item 33 9.0 7.8 1.4 5, 4, 18
100 10.3 1.5 1.6 12, 9, 10
333 9.3 0.6 1.4 10, 9, 9
1000 11.3 5.7 1.7 16, 13, 5
2500 7.7 4.0 1.2 10, 3, 10
5000 5.0 2.0 0.8 3, 5, 7
  AAC 100 135.7 40.6 20.4 106, 182, 119
TA 98 DMSO - 15.0 1.0 - 14, 16, 15
Test item 33 17.7 4.6 1.2 15, 23, 15
100 20.0 7.9 1.3 17, 14, 29
333 14.7 1.2 1.0 14, 14, 16
1000 16.3 5.7 1.1 18, 10, 21
2500 15.3 8.3 1.0 22, 6, 18
5000 9.0 1.7 0.6 8, 8, 11
  NOPD 10 537.3 23.0 35.8 514, 560, 538
E. coli DMSO - 26.7 8.1 - 21, 36, 23
Test item 33 25.0 2.0 0.9 23, 27, 25
100 27.7 4.2 1.0 23, 31, 29
333 28.0 4.6 1.1 32, 23, 29
1000 18.0 1.7 0.7 17, 17, 20
2500 16.0 5.2 0.6 13, 13, 22
5000 14.0 3.0 0.5 11, 17, 14
  4-NQO 5 717.0 107.4 26.9 841, 656, 654
Conclusions:
Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.
Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay according to OECD TG 471 and GLP.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5000 μg/plate (SPT) 33 μg - 5000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2500 μg/plate onward.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

CONCLUSION: Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 Aug 2018 - 18 Oct 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
29 Jul 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
No L142, May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Version / remarks:
Aug 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: ZH 472 redestilliert.
- Date of production: Jan 2018.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
- Stability under test conditions: The stability of the test substance under storage conditions was guaranteed until Jan 2020 as indicated by the sponsor and the sponsor holds this responsibility.
- Homogeneity: The homogeneity of the test substance was guaranteed on account of the high purity and was ensured by mixing before preparation of the test substance preparations.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed and topped up with the chosen vehicle (HAM´s F12) to achieve a concentration of the stock solution of 9.50 mg/mL. The required concentration of
1900.0 μg/mL per culture was achieved with the application of 200 μL/mL of this stock solution. To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken and pipetted thoroughly. The further concentrations were diluted from the stock solution (9.50 mg/mL) according to the planned doses. To achieve the required concentration in the test culture of the respective test substance concentrations, the application volume was 200 μL/mL. All test substance solutions were prepared immediately before administration. This study was performed in an aqueous test system. Due to the use of culture medium (Ham’s F12) as vehicle the verification of the stability of the test substance in the vehicle is not required.
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
For cell lines:
- Type and source of cells: The CHO (Chinese hamster ovary) cell line is a permanent cell line derived from the Chinese hamster.
- Absence of Mycoplasma contamination: Each batch used for mutagenicity testing was checked for mycoplasma contamination.
- Number of passages if applicable: At least 2 passages were performed before cells were taken for the experiment. A further passage was also necessary in order to prepare test cultures.
- Methods for maintenance in cell culture: Cell stocks (1.0-mL portions) stored in the gas phase above the liquified nitrogen were thawed at 37°C in a water bath. 0.5 mL of stock cultures were pipetted into 25 cm2 plastic flasks containing 5 mL Ham's F12 medium (incl. 10% [v/v] FCS). After 24 hours, the medium was replaced to remove any dead cells.
- Cell cycle length, doubling time or proliferation index : doubling time of about 12 - 16 hours.
- Modal number of chromosomes: karyotype with a modal number of 20 chromosomes.
- Periodically ‘cleansed’ of spontaneous mutants: yes. During the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
- All media were supplemented with: 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL), 1% (v/v) amphotericine B (stock solution: 250 μg/mL)
- Culture medium: Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS).
- Treatment medium (without S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10%(v/v) FCS.
- Treatment medium (with S9 mix): Ham's F12 medium containing stable glutamine and hypoxanthine.
- Pretreatment medium ("HAT" medium): Ham's F12 medium supplemented with: hypoxanthine (13.6 x 10-3 mg/mL), aminopterin (0.18 x 10-3 mg/mL), thymidine (3.88 x 10-3 mg/mL), 10% (v/v) FCS
- Selection medium ("TG" medium): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with: 6-thioguanine (10 μg/mL), 10% (v/v) FCS
- In this study, all incubations were performed at 37°C with a relative humidity of ≥ 90% in a 5% (v/v) CO2 atmosphere.
Metabolic activation:
with and without
Metabolic activation system:
co-factor-supplemented rat liver S9 (Phenobarbital/beta-Naphthoflavone induced)
Test concentrations with justification for top dose:
Test concentrations: 0, 14.8, 29.7, 59.4, 118.8, 237.5, 475.0, 950.0, 1900.0 µg/mL with and without metabolic activation.

The doses/concentrations tested in this study were selected in accordance with the requirements set forth in the test guidelines and based on the results of a preliminary range finding test.
Following the requirements of the current international guidelines and the ICPEMC Task Group a test substance should be tested up to a maximum concentration of 2 mg/mL, 2 μL/mL or 10 mM, whichever is the lowest. In this case the top concentration was based on the molecular weight of the compound (<10mM). In case of toxicity, the top dose should result in approximately 10 - 20% relative survival (adjusted cloning efficiency), but not less than 10%. For relatively insoluble test substances at least one concentration should be scored showing no precipitation in culture medium at the end of the exposure period.
In the pre-test in the absence of S9 Mix the pH value was not relevantly influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. Precipitation of the test substance in the vehicle DMSO was not observed in the stock solution (Test group: 1900.0 μg/mL). In culture medium, no test substance precipitation occurred up to the highest applied concentration in the absence and presence of S9 mix. After 4 hours treatment in the absence of S9 mix, cytotoxicity was not observed as indicated by a reduced RS of about or below 20% of control. In contrast, in the presence of S9 mix, a reduced relative RS was observed after treatment with 29.7 μg/mL and after treatment with 1900.0 μg/mL.
Vehicle / solvent:
Due to the limited solubility of the test substance in water at a concentration of 19.0 mg/mL, dimethyl sulfoxide (DMSO) was selected as vehicle in the pretest, which had been demonstrated to be suitable in the CHO/HPRT assay and for which historical control data were available.
The final concentration of the vehicle DMSO in culture medium was 1% (v/v). As requested by the sponsor, in the main experiments culture Medium (Ham’s F12) was used as vehicle. The top concentration of the stock solution was 9.5 mg/mL in culture medium (Ham’s F12) with an application volume of 200 μL/mL culture media.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF TREATMENT/ EXPOSURE: in medium.
- Cell density at seeding (if applicable): 20x10^6 cells/flask.

TREATMENT SCHEDULE:
- Preincubation period: 20-24 h.
- Exposure duration: 4 hours.
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7-9 days.
- Fixation time (start of exposure up to fixation): from day 16.

SELECTION AGENT (mutation assays): thioguanine (6-TG); 10 μg/mL

DETERMINATION OF CYTOTOXICITY
- Cloning efficiency (CE, %).
- Cell morphology and call attachment at the end of the exposure period.
Evaluation criteria:
Acceptance criteria:
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should be within our historical negative control data range (95% control limit). Weak outliers can be judged acceptable if there is no evidence that the test system is not “under control”.
• Concurrent positive controls both with and without S9 mix should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase in mutant frequencies compared with the concurrent negative/vehicle control.

Assessment criteria:
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in mutant frequencies is obtained.
• A dose-related increase in mutant frequencies is observed.
• The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative/vehicle control value and the range of our laboratory’s historical negative control data (95% control limit).

Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
• The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).
Statistics:
An appropriate statistical trend test (MS EXCEL function RGP) was performed to assess a possible dose-related increase of mutant frequencies. The used model is one of the proposed models of the International Workshop on Genotoxicity Test procedures Workgroup Report. The dependent variable was the corrected mutant frequency and the independent variable was the concentration. The trend was judged as statistically significant whenever the one-sided p-value (probability value) was below 0.05 and the slope was greater than 0. In addition, a pair-wise comparison of each test group with the vehicle control group was carried out using one-sided Fisher's exact test with Bonferroni-Holm correction. The calculation was performed using R. If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Summary of Results

Exp. Exposure period
[h]
Test groups
[μg/mL]
S9
mix
Prec.* Genotoxicity**
MFcorr.
[per 106cells]
Cytotoxicity***
RS
[%]
CE2
[%]
1 4 Negative control - n.d. 1.06 100 100
14.8 - - n.c. 85.2 n.c.
29.7 - - n.c. 88.8 n.c.
59.4 - - n.c. 81.8 n.c.
118.8 - - n.c. 66.4 n.c.
237.5 - - 2.7 77.5 88.3
475 - - 0.3 87 89.4
950 - - 1.01 74.8 79
1900 - - 2.08 66.1 76.4
Positive control1 - n.d. 222.67s 47.3 65.5
2 4 Negative control + n.d. 2.26 100 100
14.8 + - n.c. 105.2 n.c.
29.7 + - n.c. 88.8 n.c.
59.4 + - n.c. 113.8 n.c.
118.8 + - n.c. 109.2 n.c.
237.5 + - 0.92 85.5 105.5
475 + - 3.69 98.6 87.4
950 + - 2.88 84.5 89.7
1900 + - 1.44 28.3 89.4
Positive control2 + n.d. 82.19s 56.3 70.6

*        Macroscopically visible precipitation in culture medium at the end of exposure period

**       Mutant frequency MFcorr.: mutant colonies per 106 cells corrected with the CE2 value

***       Cloning efficiency related to the respective vehicle control

s     Mutant frequency statistically significant higher than corresponding control values (p ≤ 0.05)

n.c.        Culture was not continued since a minimum of only four analysable concentrations is required

n.d.        Not determined

1            EMS 400 μg/mL

2            DMBA 1.25 μg/mL

Table 2: HISTORICAL NEGATIVE CONTROL DATA, Summary (all vehicles), Period: March 2016 – December 2017

 

Corrected Mutant Frequency**

Without S9 mix - all vehicles*

With S9 mix - all vehicles*

Exposure period

4 hrs

4 hrs

Mean

2.86

2.93

Minimum

0.00

0.00

Maximum

7.09

9.93

Standard Deviation

1.81

2.24

95% Lower Control Limit

0.00

0.00

95% Upper Control Limit

6.49

7.43

No. of Experiments

69

72

* = culture medium, water 10% (v/v), DMSO 1% (v/v), acetone 1% (v/v)

** = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

Table 3: HISTORICAL POSITIVE CONTROL DATA, Summary (all experimental conditions), Period: March 2016 – December 2017

 

Corrected Mutant Frequency*

Without S9 mix

400μg/mL ethyl methanesulfonate (EMS)

With S9 mix

1.25 μg/mL 7,12-Dimethylbenz[a]anthracene (DMBA)

Exposure period

4 hrs

4 hrs

Mean

158.01

125.89

Minimum

42.47

21.52

Maximum

419.90

270.48

Standard Deviation

80.87

56.16

95% Lower Control Limit

0.00

13.02

95% Upper Control Limit

320.94

238.77

No. of Experiments

63

69

* = mutant frequency (per 1 million cells) corrected with the cloning efficiency at the end of the expression period (CE2)

Conclusions:
Thus, in the absence and the presence of metabolic activation, the test substance is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.
Executive summary:

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Two independent experiments were carried out. The 1st Experiment with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Due to technical reasons in the experimental part with S9 Mix, a repeat experiment, designated 2nd Experiment was performed. According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested. The highest tested concentration was based on the molecular weight of the compound (<10mM). Test groups printed in bold type were evaluated for gene mutations:

1st Experiment:

  • without S9 mix 0; 14.8; 29.7; 59.4; 118.8; 237.5; 475.0; 950.0; 1900.0 μg/mL
  • with S9 mix (invalid, data not shown) 0; 14.8; 29.7; 59.4; 118.8; 237.5; 475.0; 950.0; 1900.0 μg/mL

2nd Experiment

  • with S9 mix 0; 14.8; 29.7; 59.4; 118.8; 237.5; 475.0; 950.0; 1900.0 μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

In this study, in the absence and presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest concentrations evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any statistical significant and dose-dependent increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 March 2018 - 16 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
yes
Remarks:
Based on in-house non-GLP validation experiments, the treatment, the recovery phase and harvest time, was slightly modified to get distinct responses of statistical significance when using the specified positive controls.
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: ZH 472 redestilliert.
- Expiration date of the lot/batch: January 2020.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature.
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: The stability of the test substance at room temperature in the vehicle DMSO over a period of 4 hours was verified analytically. Experiment III and IV was performed in an aqueous test system. Due to the use of culture medium as vehicle, the verification of the stability of the test substance in the vehicle is not required. All formulations were prepared freshly before treatment and used within two hours of preparation.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Stock formulations of the test item and serial dilutions were made in DMSO (exp. I and II) or culture medium (exp. III and IV). The final concentration of DMSO in the culture medium was 0.5 %. The solvents were chosen due to their solubility properties and relative non-toxicity to the cell cultures. All formulations were prepared freshly before treatment and used within two hours of preparation.
Due to the solubility properties of the test item in aqueous media and due to customer request, all experimental parts (4 hour treatment in the absence and presence of S9 mix and continuous treatment in the absence of S9 mix) were repeated by using a different solvent (culture medium).
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Human lymphocytes.
- Suitability of cells: Yes. Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.
- Normal cell cycle time (negative control):

For cell lines:
- Absence of Mycoplasma contamination:
- Number of passages if applicable:
- Methods for maintenance in cell culture:
- Cell cycle length, doubling time or proliferation index :
- Modal number of chromosomes:
- Periodically checked for karyotype stability: [yes/no]
- Periodically ‘cleansed’ of spontaneous mutants: [yes/no]

For lymphocytes:
- Sex, age and number of blood donors: Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a female donor (31 years old) for Experiment I, from a female donor (35 years old) for Experiment II, from a male donor (21 years old) for Experiment III and from a female donor (28 years old) for Experiment IV. The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Whether whole blood or separated lymphocytes were used: whole blood.
- Whether blood from different donors were pooled or not: no.
- Mitogen used for lymphocytes: Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.
Metabolic activation:
with and without
Metabolic activation system:
co-factor-supplemented rat liver S9 (Phenobarbital/β-Naphthoflavone induced)
Test concentrations with justification for top dose:
Experiment I: 14.3; 25.0; 43.7; 76.4; 134; 234, 410; 717; 1255; 1882 µg/mL (with/without S9)
Experiment II: 76.4; 134; 234; 410; 717; 1255; 1882 µg/mL (without)
Experiment III: 65.5; 115; 201; 351; 615; 1075; 1882 µg/mL (with/without S9)
Experiment IV: 134; 234; 410; 717; 1255; 1882 µg/mL (without)

Dose selection was performed according to the current OECD Guideline for the in vitro micronucleus test. If no precipitate or limiting cytotoxicity is observed, the highest test concentration should correspond to 10 mM, 2 mg/mL or 2 μl/mL, whichever is the lowest. At least three test item concentrations should be evaluated for cytogenetic damage.
In case of test item induced cytotoxicity, measured by a reduced cytokinesis-block proliferation index (CBPI) and expressed as cytostasis, or precipitation the dose selection should reflect these properties of the test item. Where cytotoxicity occurs, the applied concentrations should cover a range from no to approximately 55 ± 5 % cytostasis.
With regard to the molecular weight of the test item, 1882 μg/mL (approx. 10 mM) were applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations ranging from 14.3 to 1882 μg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test for toxicity, no precipitation of the test item was observed. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Since no cytotoxic effects were observed, 1882 μg/mL were chosen as top treatment concentration in all further experiments.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium with 0.5 % DMSO (exp. I and II) or culture medium (exp. III and IV).

- Justification for choice of solvent/vehicle:

- Justification for percentage of solvent in the final culture medium:
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
culture medium with 0.5 % DMSO (exp. I and II) or culture medium (exp. III and IV)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: demecolcine, without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 h incubation with PHA (mitogen) to stimulate lymphocyte proliferation
- Exposure duration: 4 h (pulse exposure); 20 h (continuous exposure; without S9 mix)
- Expression time (cells in growth medium): 16 h (recovery period; pulse exposure only) + another 20 h after addition of Cytochalasin B
- Fixation time (start of exposure up to fixation of cells): 40 hours

SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin B (4 μg/mL)

STAIN (for cytogentic assay): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
- cultures were harvested by centrifugation (5 min)
- cells were resuspended in approximately 5 mL "saline G" and spun down once again (5 min)
- cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes
- 1mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added and the cells were resuspended
- after removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold
- slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide
- cells were stained with Giemsa.

NUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976).
- The micronuclei have to be stained in the same way as the main nucleus.
- The area of the micronucleus should not extend the third part of the area of the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: determination of CBPI (Cytokinesis-block proliferation index) in 500 cells per culture; A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
CBPI = ((Mononucleate cells x 1) + (Binucleate cells x 2) + (Multinucleate cells x 3)) / Total number of cells
Evaluation criteria:
A test item can be classified as non-clastogenic and non-aneugenic if:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval).

test item can be classified as clastogenic and aneugenic if:
− At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− The increase is concentration-related in at least one experimental condition
− The results are outside the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval).
Statistics:
Statistical significance was confirmed by the Chi square test (α < 0.05), using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis was conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.
A linear regression was performed using a validated test script of "R", a language and environment for statistical computing and graphics, to assess a possible dose dependency in the rates of micronucleated cells. The number of micronucleated cells obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Both, biological and statistical significance were considered together.
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid

Table 1: Summary of results

Exp.

Preparation

interval

Test item

concentration

in µg/mL

Proliferation

index

CBPI

Cytostasis

in %*

Micronucleatedcells in %**

95% Ctrl limit

Exposure period 4 hrs without S9 mix

I

40 hrs

Solvent control1

1.61

 

0.55

0.06 – 1.19

Positive control3

1.40

33.3

13.35S

3.92 – 25.34

717

1.41

32.8

0.95

 

1255#

1.47

23.1

1.55S

 

1882

1.44

26.9

0.90

 

III

40 hrs

Solvent control2

1.76

 

0.55

0.06 – 1.19

Positive control3

1.66

14.1

13.35S

3.92 – 25.34

615

1.71

7.5

0.65

 

1075

1.65

14.4

0.45

 

1882

1.67

12.0

0.65

 

Exposure period 20 hrs without S9 mix

II

40 hrs

Solvent control1

1.37

 

0.40

0.00 – 1.11

Positive control4

1.18

52.0

2.90S

1.47 – 5.89

717

1.38

n.c.

1.35S

 

1255

1.30

18.8

0.25

 

1882PS

1.23

38.9

1.10S

 

IV

40 hrs

Solvent control2

1.97

 

0.80

0.00 – 1.11

Positive control5

1.87

10.1

2.70S

1.47 – 5.89

717

1.90

6.8

0.45

 

1255

1.93

3.7

0.80

 

1882

1.96

0.6

0.60

 

Exposure period 4 hrs with S9 mix

I

40 hrs

Solvent control1

1.57

 

0.35

0.08 – 1.38

Positive control6

1.32

43.3

3.90S

0.70 – 10.20

717

1.55

3.2

0.35

 

1255

1.48

15.5

0.85S

 

1882

1.61

n.c.

0.30

 

III

40 hrs

Solvent control2

1.77

 

0.35

0.08 – 1.38

Positive control7

1.62

19.3

3.20S

0.70 – 10.20

615

1.87

n.c.

0.45

 

1075

1.82

n.c.

0.55

 

1882

1.89

n.c.

0.50

 

* For the positive control groups and the test item treatment groups the values are related to the solvent controls

** The number of micronucleated cells was determined in a sample of 2000 binucleated cells

# The number of micronucleated cells was determined in a sample of 4000 binucleated cells

S The number of micronucleated cells is statistically significantly higher than corresponding control values

PS Phase separation occurred at the end of treatment

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

1 DMSO 0.5 % (v/v)

2 Culture medium 100 % (v/v)

3 MMC 0.8 μg/mL

4 Demecolcine 150 ng/mL

5 Demecolcine 50 ng/mL

6 CPA 15.0 μg/mL

7 CPA 17.5 μg/mL

Table 2: Historical laboratory control data, Percentage of micronucleated cells in human lymphocyte cultures (2017)

Solvent Control 
  without S9 with S9
Pulse treatment (4/40) Continuous treatment (20/40) Pulse treatment (4/40)
No. of experiments 61* 65** 80***
Mean 0.62 0.56 0.73
95 % Ctrl limit 0.06 – 1.19 0.00 – 1.11 0.08 – 1.38
1x SD (2x SD) 0.28 (0.56) 0.28 (0.56) 0.33 (0.66)
Min – Max 0.00 – 1.18 0.05 – 1.10 0.10 – 1.85
Positive Control 
  without S9 with S9
Pulse treatment (4/40) Continuous treatment (20/40) Pulse treatment (4/40)
Substance MMC Demecolcine CPA
No. of experiments 62 65 86
Mean 14.63 3.68 5.45
95 % Ctrl limit 3.92 – 25.34 1.47 – 5.89 0.70 – 10.20
1x SD (2x SD) 5.35 (10.7) 1.11 (2.22) 2.37 (4.74)
Min – Max 2.60 – 28.50 2.10 – 8.80 2.25 – 13.30

* Aqueous solvents – 21 Experiments; Organic solvents – 40 Experiments

** Aqueous solvents – 24 Experiments; Organic solvents – 41 Experiments

*** Aqueous solvents – 30 Experiments; Organic solvents – 50 Experiments

Conclusions:
Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.
Executive summary:

The test item, dissolved in DMSO (exp. I and II) or culture medium (exp. II and IV), was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.

Four independent experiments were performed. In Experiment I and Experiment III, the exposure period was 4 hours with and without S9 mix. In Experiment II and Experiment IV, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analyzed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (1882 μg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.

In Experiment I, Experiment III and Experiment IV in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence of S9 mix, moderate cytotoxicity was observed at the highest applied concentration, which showed phase separation.

In Experiment I in the presence of S9 mix, one statistically significant increase (0.85% micronucleated cells) was observed after treatment with 1255 μg/mL of the test item. Since the value is clearly within the historical control data range of 0.08 – 1.38 % micronucleated cells and no dose-dependency tested via trend test could be observed, this finding can be regarded as biologically irrelevant.

In Experiment I in the absence of S9 mix, one statistically significant increase (1.55% micronucleated cells), clearly above the historical control data range of 0.06 – 1.19% micronucleated cells was observed after treatment with 1255 μg/mL of the test item. No dose-dependency, tested via trend test could be observed.

In Experiment II in the absence of S9 mix, statistically significant increases (1.35 and 1.10% micronucleated cells) were observed after continuous treatment with 717 and 1882 μg/mL of the test item. The value of 1.35 % clearly exceeded the range of the historical control data (0.00 – 1.11 % micronucleated cells). No dose-dependency, tested via trend test could be observed.

In Experiment III in the absence and presence of S9 mix and in Experiment IV in the absence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro:

Bacterial Reverse Mutation Assay (Ames Test)

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay according to OECD TG 471 and GLP.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 33 μg - 5000 μg/plate (SPT) 33 μg - 5000 μg/plate (PIT)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: A weak bacteriotoxic effect was occasionally observed depending on the strain and test conditions from about 2500 μg/plate onward.

MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test or in the preincubation test without S9 mix or after the addition of a metabolizing system.

CONCLUSION: Under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

HPRT gene mutation assay

The test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro according to OECD TG 476 and GLP. Two independent experiments were carried out. The 1st Experiment with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Due to technical reasons in the experimental part with S9 Mix, a repeat experiment, designated 2nd Experiment was performed. According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following concentrations were tested. The highest tested concentration was based on the molecular weight of the compound (<10mM). Test groups printed in bold type were evaluated for gene mutations:

1st Experiment:

  • without S9 mix 0; 14.8; 29.7; 59.4; 118.8;237.5; 475.0; 950.0; 1900.0μg/mL
  • with S9 mix (invalid, data not shown) 0; 14.8; 29.7; 59.4; 118.8; 237.5; 475.0; 950.0; 1900.0 μg/mL

2nd Experiment

  • with S9 mix 0; 14.8; 29.7; 59.4; 118.8;237.5; 475.0; 950.0; 1900.0μg/mL

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]-anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations.

In this study, in the absence and presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest concentrations evaluated for gene mutations.

Based on the results of the present study, the test substance did not cause any statistical significant and dose-dependent increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system.

Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Micronucleus Test In Human Lymphocytes

The induction of cytogenetic damage in human lymphocytes was assessed by means of a Micronucleus Test In Human Lymphocytes according to OECD TG 487 and GLP.

The test item, dissolved in DMSO (exp. I and II) or culture medium (exp. II and IV), was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix.

Four independent experiments were performed. In Experiment I and Experiment III, the exposure period was 4 hours with and without S9 mix. In Experiment II and Experiment IV, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analyzed. Per culture at least 1000 binucleated cells were evaluated for cytogenetic damage.

The highest applied concentration in this study (1882 μg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487.

Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.

In Experiment I, Experiment III and Experiment IV in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence of S9 mix, moderate cytotoxicity was observed at the highest applied concentration, which showed phase separation.

In Experiment I in the presence of S9 mix, one statistically significant increase (0.85% micronucleated cells) was observed after treatment with 1255 μg/mL of the test item. Since the value is clearly within the historical control data range of 0.08 – 1.38 % micronucleated cells and no dose-dependency tested via trend test could be observed, this finding can be regarded as biologically irrelevant.

In Experiment I in the absence of S9 mix, one statistically significant increase (1.55% micronucleated cells), clearly above the historical control data range of 0.06 – 1.19% micronucleated cells was observed after treatment with 1255 μg/mL of the test item. No dose-dependency, tested via trend test could be observed.

In Experiment II in the absence of S9 mix, statistically significant increases (1.35 and 1.10% micronucleated cells) were observed after continuous treatment with 717 and 1882 μg/mL of the test item. The value of 1.35 % clearly exceeded the range of the historical control data (0.00 – 1.11 % micronucleated cells). No dose-dependency, tested via trend test could be observed.

In Experiment III in the absence and presence of S9 mix and in Experiment IV in the absence of S9 mix, no relevant increases in the numbers of micronucleated cells were observed after treatment with the test item.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test substance is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.