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Genetic toxicity in vitro

Description of key information

The test substance was tested in an Ames test at concentrations of 1.5 to 1500 ug/plate (cytotoxicity observed) in the salmonella strains TA98, TA100, TA1535 and TA1537 (Harlan 2013). The concentrations in the test on E. coli WP2 uvr A were 15 -5000 ug/plate. At 1500 and 5000 ug/plate a greasy precipitate was observed, that did not hamper the evaluation. In a plate incorporation and a pre-incubation assay (both performed in triplicate) with and without metabolic activation, the test substance did not induce mutations (base-pair substitution and frame-shift type) in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, as well as in E. coli WP2 uvr. The test substance was tested in a chromosome aberration assay in human lymphocytes according to OECD 473 (1997) (Harlan 2015). Duplicate cultures were exposed for 4 (with and without metabolic activation) and 24 hours (without metabolic activation) with a 20 h expression time in two separate experiments. Heamolysis and/or precipitate was observed at concentrations of 78 ug/mL and above. Scoring for aberrations revealed no increases in the number of cells with aberrations. In addition no increased number of polyploid cells was observed in any of the treatments. Vehicle and positive controls were within expected (historical) ranges. The test substance is considered to be non-clastogenic to human lymphocytes in vitro. The test substance was tested in L5178Y cells in presence and absence of metabolic activation in a study performed according to OECD 476. In both experiments the test substance did not induce an increase in mutant frequency at the TK +/- locus and is therefore considered to be non-mutagenic.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16-01-2013 to 19-03-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to the guidelines, under GLP
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium: histidine
E-coli: 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 from rats induced with Phenobarbitone/ß-Naphthoflavone
Test concentrations with justification for top dose:
Salmonella species: 1.5, 5, 15, 50, 150, 500 and 1500 ug/plate with and without metabolic activation in preincubation and plate incorporation assay
E. coli: 50, 150, 500, 1500 and 5000 ug/plate with and without metabolic activation in plate incorporation assay; 15, 50, 150, 500, 1500 and 5000 ug/plate with and without metabolic activation in preincubation assay
Vehicle / solvent:
acetone (100 mg/mL)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
for TA100, TA1535 and WP2uvrA without metabolic activation
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
for TA1537 without metabolic activation
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
for TA100, TA1535, TA 1537 and WP2uvrA with metabolic activation
Positive control substance:
other: 2-aminoanthracene
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
for TA98 without metabolic acivation
Positive control substance:
4-nitroquinoline-N-oxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Remarks:
for TA98 with metabolic activation
Positive control substance:
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: 1st test in agar (plate incorporation); 2nd test preincubation

DURATION
- Preincubation period: 20 min at 37°C
- Exposure duration: 48 hours at 37°C

NUMBER OF REPLICATIONS: 3/concentration

DETERMINATION OF CYTOTOXICITY
- Method: growth rate + presence of bacterial background lawn

COLONY COUNT:
Colony counter

Evaluation criteria:
A test item will be considered mutagenic (positive) in the test system if one or more of these criteria are met
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).
Statistics:
Not specified according to UKEMS
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduction of bacterial background lawn at 1500 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
presence of greasy precipitate did not hinder the evaluation at 1500 and 5000 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Conclusions:
Interpretation of results
negative without metabolic activation
negative with metabolic activation

The test substance did not induce mutations (base-pair substitution and frame-shift type) in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 as well as in E. coli WP2 uvr A both in presence and absence of metabolic activation.
Executive summary:

The test substance was tested in an Ames test at concentrations of 1.5 to 1500 ug/plate (cytotoxicity obeserved) in the salmonella strains TA98, TA100, TA1535 and TA1537. The concentrations in the test on E. coli WP2 uvr A were 15 -5000 ug/plate. At 1500 and 5000 ug/plate a greasy precipitate was observed, that did not hamper the evaluation. In a plate incorporation and a pre-incubation assay (both performed in triplicate) with and without metabolic activation, the test substance did not induce mutations (base-pair substitution and frame-shift type) in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537, as well as in E. coli WP2 uvr.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-12-2014 to 10-04-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline study under GLP, the study follows OECD 473 1997
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: fresh heparinized whole human blood (AGT ca 16 h)
Details on mammalian cell type (if applicable):
- Type and identity of media:
culture medium: MEM, supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% foetal bovine serum (FBS)
test medium: MEM, supplemented with test substance (0.05 mL) or vehicle (0.05 mL) or positive control (0.1 mL) and 1-2% S9 mix (tests with metabolic activation)
before the test lymphocytes were stimulated to divide by the addition of phytohaemagglutinin (PHA).

- Properly maintained: yes/no
- Periodically checked for Mycoplasma contamination: yes/no
- Periodically checked for karyotype stability: yes/no
- Periodically "cleansed" against high spontaneous background: yes/no
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
prepared from in-house from male rats induced with Phenobarbitone/β-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4.
Test concentrations with justification for top dose:
dose range test: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 µg/mL
1st and 2nd experiment:
without S9: 0, 5, 10, 20, 40, 60 and 80 µg/mL (evaluation 1st exp 20, 40 and 60 µg/mL; 2nd exp 10, 20 and 40 ug/mL)
with S9-mix (1-2%): 0, 10, 20, 40, 60, 80 and 160 µg/mL ((evaluation 1st exp 40, 60 and 80 µg/mL; 2nd exp 20, 40 and 60 ug/mL)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: substance was soluble at 500 mg/mL in acetone (not soluble in DMSO)
- maximum tested concentration in cytotoxicity test 2500 ug/mL (100 uL acetone, because human lymphocytes are sensitive to acetone in higher amounts)
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Remarks:
cyclophophamide with metabolic activation, Mitomycin C without metabolic activation
Details on test system and experimental conditions:
DURATION
- Exposure duration: 4 h without S9, 4 h with S9, 24 h without S9
- Expression time (cells in growth medium): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): colcemid (2 hours before harvest)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2 per treatment

NUMBER OF CELLS EVALUATED: Where possible the first 100 consecutive well-spread metaphases from each culture were
counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. In the first experiment the number of metaphases counted for the MCC (0.4 ug/mL) control was 60 (with aberrations in 53% and 37% of the cells)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Evaluation criteria:
Biological relevance will be taken into account first

Negative Control: The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control
cultures will normally be within the laboratory historical control data range.

Positive Control: All the positive control chemicals must induce a clear positive response (p≤0.01). Acceptable
positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.

non-genotoxic if:
1. The number of induced chromosome aberrations in all evaluated dose groups is within the range of laboratory historical control data.
2. No toxicologically or statistically significant increase of the number of structural chromosome aberrations is observed following statistical analysis.

genotoxic if:
1. The number of induced structural chromosome aberrations is not in the range of laboratory historical control data, and
2. Either a concentration-related or a statistically significant increase of the number of structural chromosome aberrations is observed. Marked increases only observed in one dose level will be assessed on a case by case basis.
Statistics:
NA
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
4 h without/with S9: precipitate and haemolysis at 78 ug/mL and above; 24 h without S9: precipitate at and above 78 ug/mL
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none (pH 7.31-7.43)
- Effects of osmolality: none (339-378 mOsm)
- Precipitation: at 78 ug/L and above
Conclusions:
Interpretation of results
negative without metabolic activation
negative with metabolic activation

The substance does not induce chromosome aberrations in presence and absence of metabolic activation and is considered non-clastogenic.
Executive summary:

The test substance was tested in a chromosome aberration assay in human lymphocytes according to OECD 473 (1997). Duplicate cultures were exposed for 4 (with and without metabolic activation) and 24 hours (without metabolic activation) with a 20 h expression time in two separate experiments. Heamolysis and/or precipitate was observed at concentrations of 78 ug/mL and above. Scoring for aberrations revealed no increases in the number of cells with aberrations. In addition no increased number of polyploid cells was observed in any of the treatments. Vehicle and positive controls were within expected (historical) ranges.

The test substance is considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Dec 2014 to 17 Feb 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test according to the guideline and under GLP
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase, TK +/-, locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Cells obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.

Culture medium: R10
RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/ml), Streptomycin (100 µg/ml), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/ml) and 10% donor horse serum at in air at 37 °C with 5% CO2.

Medium: R20/R0
RPMI 1640 with 20% donor horse serum/RPMI 1640

A cleaning procedure to remove homozygous (TK -/-) mutants was applied
Additional strain / cell type characteristics:
other: generation time ca. 12 hours
Metabolic activation:
with and without
Metabolic activation system:
(S9) phenobarbital/β-naphthoflavone induced rat liver extract
Test concentrations with justification for top dose:
Preliminary toxicity test:
without S9 (4 and 24 h incubation), with S9 (4 h incubation: 0 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 ug/mL (pH 7.31-7.43; osmolality 339-378 mOsm)

Experiment 1 (values in brackets not scored)
without S9 (4 h incubation): 0, (0.63), 1.25, 2.5, 5, 10, 20, 30 and (40) ug/mL
with 2% S9 (4 h incubation): 0, 10, 20, 30, 40, 50, (60), (70) and (80) ug/mL

Experiment 2 (values in brackets not scored)
without S9 (24 h incubation): 0, 1.25, 2.5, 5, 10, 15, 20, (25), (30), (35) and (40) ug/mL
with 1% S9 (4 h incubation): 0, 10, 20, 25, 30, 35, 40, (45), (50), (55) and (60) ug/mL
Vehicle / solvent:
acetone (concentration in test medium < 0.5%)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Remarks:
EMS in absence of metabolic activation; CP in presence of metabolic activation
Details on test system and experimental conditions:
DURATION
- Exposure duration: 4 h (with and without S9); 24 h (without S9) incubated at 37°C with continuous shaking
- Expression time (cells in growth medium): incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days, thereafter cells were plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates.
- Scoring after 10 to 14 days incubation in microwell plates at 37 °C with 5% CO2 in air

STAIN: MTT

NUMBER OF REPLICATIONS: 2/concentration, 4 for vehicle controls

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth + viability -->relative total growth,

Evaluation criteria:
a statistically sugnificant increase of the induced mutant frequency over the vehicle mutant frequency (reaction should be above the Global Evaluation Factor of 1.26E-06 and has to show a positive trend)
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity without S9: at 39.06 ug/mL and above; with S9: at 78.13 ug/mL and above as measured in the preliminary test
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In experiment 1 cytotoxicity was very slightly below the optimum level of toxicity in the experimen with metabolic activation.

negative controls: acceptable range of 50 to 170 x 10-6 viable cells
positive controls: acceptable when observed mutant frequency is at least three to five fold increased compared to vehicle control

Precipitate was observed at and above above 78.13 ug/ml in the presence and absence of metabolic activation in the pre-test

experiment 1; no statistically significant or dose related (linear-trend) increase in the mutant frequency x 10-6 per viable cell in the absence or presence of metabolic activation. Cytotoxicity was observed at the highest evaluated concentration. Precipitate was observed at 70 ug/ml and above in the test with metabolic activation.

experiment 2; no statistically significant or dose related (linear-trend) increase in the mutant frequency x 10-6 per viable cell in the absence or presence of metabolic activation. Cytotoxicity was observed at the highest evaluated concentration. Precipitate was

observed at 50 ug/ml and above in the test with metabolic activation.

Conclusions:
Interpretation of results
negative with metabolic activation
negative without metabolic activation

The test substance did not induce an increase in mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non-mutagenic.
Executive summary:

The test substance was tested in L5178Y cells in presence and absence of metabolic activation in a study performed according to OECD 476. In both experiments the test substance did not induce an increase in mutant frequency at the TK +/- locus and is therefore considered to be non-mutagenic.

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

Genetic toxicity in vivo

Description of key information

No in vivo studies are available. No effects on mutagenicity were found in bacteria and mammalian cells. In addition no effects on chromosomes were found in human lymphocytes. Based on the outcome of these in vitro assays, no in vivo testing is considered necessary.

Additional information

Justification for selection of genetic toxicity endpoint
guideline studies under GLP

Justification for classification or non-classification

The available data on genetic toxicity of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.