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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In an Ames test the substance was tested in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli strain WP2uvrA in presence and absence of metabolic activation. No increase in mutant frequency was reported in any of the strains tested both in presence and absence of metabolic activation. The substance is considered to be non-mutagenic under the conditions of this test.

The substance was tested in the L5178Y TK +/- Mouse  Lymphoma Assay according to OECD 490 up to precipitation level. Exposure was either 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). No increase in the mutant frequency at the TK +/- locus was reported (evaluation with the Global Evaluation Factor (GEF) of 126 E-06). Therefore it is concluded that the substance is non-mutagenic in this assay.

Duplicate cultures of human lymphocytes, treated with the substance, were evaluated for chromosome aberrations at up to four dose levels (maximum dose 160 mg/mL, lowest precipitating level), together with vehicle and positive controls in presence of metabolic activation (4 hours) and in absence of metabolic activation (4 and 24 hours).  The substance did not demonstrate any marked toxicity and did not induce any statistically significant increases in the frequency of cells with aberrations and was considered to be non-clastogenic to human lymphocytes in vitro.

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:
14 June 2017 to 06 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine/tryptophan
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: University of California, Berkeley, on culture discs, on 04 August 1995, • British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.
- Methods for maintenance in cell culture if applicable: stored at -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34

Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/beta-naphthaflavone induced rat liver S9 (Lot No. PB/NF S9 30 June 2017 )
Test concentrations with justification for top dose:
Test 1 (plate incorporation): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Test 2 (pre-incubation): 15, 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: substance is insoluble in water and DMSO, completely soluble in acetone at 100 mg/L
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone (in pre-incubation test 0.05 mL in final test solution)
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: exp 1 plate incorporation; exp 2 preincubation

DURATION
- Preincubation period: 30 min at 37 ± 3 °C
- Exposure duration: 48 h at 37 ± 3 °C

NUMBER OF REPLICATIONS: 3/concentration

DETERMINATION OF CYTOTOXICITY
- Method: visible reduction in the growth of the bacterial background lawn
Evaluation criteria:
a substance is considered positive when:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
Statistics:
Dunnetts Regression Analysis
Key result
Species / strain:
other: TA1535, TA1537, TA98, TA100 and WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: at 1500 and 5000 ug/plate a slight film of the substance was seen (not preventing scoring)
Conclusions:
Based on the findings in this test the substance is considered non-mutagenic in bacteria
Executive summary:

In an Ames test the substance was tested in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and E. coli strain WP2uvrA in presence and absence of metabolic activation. No increase in mutant frequency was reported in any of the strains tested both in presence and absence of metabolic activation. The substance is considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 August 2017 to 17 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy Trade and Industry (METI), and Ministry of the Environmental (MOE) Guidelines of 31 March 2011
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
NA
Species / strain / cell type:
lymphocytes: human peripheral lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: human peripheral lymphocytes
- Suitability of cells: standard cells (checked for exposure to high levels of radiation or hazardous chemicals and not knowingly recently suffered from a viral infection)
- Cell cycle length, doubling time or proliferation index: AGT 16 hours
- Sex, age and number of blood donors if applicable: Preliminary Toxicity Test: female, aged 30 years; Main Experiment: female, aged 28 years
- Whether whole blood or separated lymphocytes were used if applicable: fresh heparinized whole blood
- Methods for maintenance in cell culture: grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air
- Modal number of chromosomes: 44-48
- Normal (negative control) cell cycle time: 16 hours

MEDIA USED
- Type and identity of media including CO2 concentration: MEM at approximately 37 ºC with 5 % CO2
- Properly maintained: yes
Cytokinesis block (if used):
demecolcine (Colcemid 0.1 µg/mL) 2.5 hours before the required harvest time.
Metabolic activation:
with and without
Metabolic activation system:
S9 from rat livers induced with phenobarbital/beta-naphthoflavone
Test concentrations with justification for top dose:
Exposure Group Final concentration of

4(20)-hour without S9 0*, 5, 10, 20*, 40*, 80*, 160, MMC 0.4*
4(20)-hour with S9 (2%) 0*, 5, 10, 20*, 40*, 80*, 160, CP 4*
24-hour without S9 0*, 5, 10, 20*, 40*, 80*, 160, MMC 0.1*


* Dose levels selected for metaphase analysis
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone (99.75 % in 505 µl aliquots; maximum achieved dosing concentration 2500 µg/mL)
- Justification for choice of solvent/vehicle: insoluble in culture medium at 50 mg/mL and dimethyl sulphoxide at 500 mg/mL but soluble in acetone at 500 mg/mL
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
DURATION
- pre-incubation: 48 hours at approximately 37 ºC, 5% CO2 in humidified air
- Exposure duration: 4 hour with and without metabolic activation, 24 hours without metaboliv activation at approximately 37 ºC, 5% CO2 in humidified air
- Expression time (cells in growth medium): 4 hour tests: 20 hours at approximately 37 ºC, 5% CO2 in humidified air

SPINDLE INHIBITOR: Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) 2.5 hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2/concentration

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

NUMBER OF CELLS EVALUATED: 2000/concentration for mitotic index

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE : 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: cells with 69 chromosomes or more were scored as polyploid cells
- Determination of endoreplication: recorded separately and included in the polyplod cell total
Rationale for test conditions:
standard according to the guidance
Evaluation criteria:
•The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range
•All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix
•The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
•The required number of cells and concentrations were analyzed
Statistics:
Fisher's Exact test (p<0.05)
Key result
Species / strain:
lymphocytes: human peripheral lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 7.18 -7.32
- Effects of osmolality: 309 -347 mOs
- Evaporation from medium: none
- Water solubility: see vehicle selection
- Precipitation: yes at 80 ug/mL in all tests

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: available (all findings were within historical control values)
- Negative (solvent/vehicle) historical control data: available (all findings were within historical control values)

ADDITIONAL INFORMATION ON CYTOTOXICITY: none (29% decrease of mitotic index in 24 h test at 80 ug/mL)

details see attached document

Conclusions:
The substance is considered not clastogenic under the conditions of the test
Executive summary:

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated;4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by precipitate. The dose levels selected for the Main Test were as follows:

Group

Final concentration of substance evaluated

4(20)-hour without S9

0, 5,10, 20, 40, 80, 160

4(20)-hour with S9 (2%)

0, 5,10, 20, 40, 80, 160

24-hour without S9

0, 5,10, 20, 40, 80, 160

All vehicle (Acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not demonstrate any marked toxicity and did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level. 

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

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 November 2017 to 19 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED: L5178Y TK+/- 3.7.2c mouse lymphoma cell line
- Source of cells: MRC Cell Mutation Unit at the University of Sussex, Brighton, UK
- Cell cycle length:ca 12 hours
- Methods for maintenance in cell culture if applicable: stored in liquid nitrogen at approximately -196°C

MEDIA USED
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes, before freezing
Additional strain / cell type characteristics:
other: TK+/-
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital/beta-naphthaflavone induced rat liver S9
Test concentrations with justification for top dose:
4 h and 24 h exposure: 2.5, 5, 10, 20, 40 and 80 µg/mL

Top dose based on presence of precipitate
Vehicle / solvent:
acetone (acetone is toxic to cells at 0.5% of total volume, therefore the substance was formulated at 500 mg/mL and dosed at 0.5% to give the maximum achievable dose level of 2500 µg/mL to be used in the pre-test).
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
A pretest was conducted on cell cultures at 5 x 10 E5 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10E5 cells/mL using a 24-hour exposure period without S9. The dose range was set at 1.25 to 320 µg/mL. After exposure cells were washed twice, resuspended and counted. The cultures were serially diluted to 2 x 10E5 cells/mL. and incubated at 37 C with 5% CO2 in air and sub-cultured after 24 hours. After a further 24 hours the cultures were counted to assess Suspension Growth (SG)

In the main study exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) in R0 or R10 medium (total volume to 20 mL). The solutions were incubated at 37°C for 4 or 24 hours with continuous shaking. the initial cell number was 1 x 10E6 cells/mL for 4 hour exposures and 0.3 x 10E6 cells/mL for 24 hour exposure.
At the end of the treatment period, for each experiment, the cells were washed twice, resuspended in R20 medium and incubated at 37°C with 5% CO2 (subcultured and counted every 24 hours -> Relative Suspension Growth (%RSG)) for 2 days.

On Day 2 of the experiment, the cells were counted again, diluted to 10E4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates.On this dayadditional cells were diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.
Microtitre plates were scored using a magnifying mirror box after ten to twelve days incubation at 37°C with 5% CO2 in air. These plates were also scored for the presence of large and small colonies.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
Statistics:
Mutant 240C by York Electronic Research, which follows the statistical guidelines recommended by the UKEMS (Robinson W D et al., 1989).
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no (7.23-7.33)
- Effects of osmolality: no (277-287 mOs)
- Precipitation:
main study: onset at 80 µg/mL in all cultures

RANGE-FINDING/SCREENING STUDIES: no cytotoxicity, onset of precipitate at at 40 µg/mL in the 4-hour culture with metabolic activation and the 24 h culture and at 80 µg/mL in the 4-hour culture without metabolic activation

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) are reported in an annex to the report
Remarks on result:
other: 4 hour exposure

Treatment

(µg/ml)

4-hours -S-9 Treatment

 

Treatment

(µg/ml)

4-hours +S-9 Treatment

 

 

Treatment

(µg/ml)

 

24-hours -S-9

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

%RSG

RTG

MF§

 

 

 

%RSG

RTG

MF§

 

 

 

%RSG

RTG

MF§

0 

 

100

1.00

143.34 

 

0 

 

100

1.00

85.13 

 

0

 

100 

1.00

204.05 

0.63

Ø

94   

 

 

 

1.25

Ø

99  

 

 

 

1.25

Ø

98  

 

 

1.25

Ø

91   

 

 

 

2.5 

 

89

0.88

100.48 

 

2.5 

 

104

1.19

192.91

2.5 

 

89

0.85

162.50 

 

5 

 

90

0.98

94.87

 

5 

 

116

1.18

210.26

5 

 

75

0.67

168.38 

 

10 

 

96

1.13

97.74

 

10 

 

172

1.79

181.74

10 

 

67

0.65

154.48 

 

20 

 

83

1.25

63.85

 

20 

 

159

1.68

174.76

20 

 

76

0.70

177.80 

 

40 

 

84

1.33

63.71

 

40 

 

146

1.69

179.75

40 

 

73

0.68

174.80 

 

80 

 

97

1.12

93.82

 

80 

 

135

1.66

168.08 

80 

 

70

0.81

132.98 

 

160

Ø

90   

 

 

 

160

Ø

111  

 

 

 

MF threshold for a positive response = 269.34

 

 

MF threshold for a positive response = 211.13

 

 

MF threshold for a positive response = 330.05

EMS

 

 

 

 

 

CP

 

 

 

 

 

EMS

 

 

 

 

400

 

57

0.48

1094.30

 

1.5

 

73

0.48

880.23

 

150

 

79

0.60

1847.41

     § Positive wells per tray, 96 wells plated

 

Proportion small colony mutants

Treatment

(µg/ml)

4-hours -S-9 Treatment

4-hours +S-9 Treatment

 

24-hours -S-9

0 

0.43

0.35

0.53

2.5 

0.48

0.54

0.54

5 

0.43

0.33

0.48

10 

0.44

0.24

0.54

20 

0.45

0.30

0.36

40 

0.44

0.47

0.49

80 

0.47

0.38

0.39

Positive control

0.50

0.84

0.51

 

Conclusions:
The substance is considered non-mutagenic in the L5178Y TK +/- Mouse Lymphoma Assay
Executive summary:

The substance was tested in the L5178Y TK +/- Mouse  Lymphoma Assay according to OECD 490 up to precipitation level. Exposure was either 4 hours (with and without metabolic activation) or 24 hours (without metabolic activation). No increase in the mutant frequency at the TK +/- locus was reported (evaluation with the Global Evaluation Factor (GEF) of 126 E-06). Therefore it is concluded that the substance is non-mutagenic in this assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the available information no classification for mutagenicity is necessary according to Regulation (EC) No 1272/2008 (CLP).