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Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation assay: The test substance was not mutagenic in the bacterial reverse mutation assay, according to OECD 471.

HPRT assay: The test substance did not induce gene mutation in the HPRT assay, according to OECD 476.

Micronuclei: The test substance was concluded to be negative for the induction of micronuclei in both non-acitvated and S9 -activated test systems in the in vitro micronucleus test, according to OECD 487.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-03-16 to 2001-04-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix, Aroclor 1254 induced
Test concentrations with justification for top dose:
experiment 1: 0, 50, 150, 500, 1500, 5000 µg/plate
experiment 2: 0, 5, 15, 50, 150, 500, 1500 µg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
mitomycin C
other: with S9 mix: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth



Evaluation criteria:
The number of spontaneous revertants observed using each of the five strains was close to those previously established in our laboratory and was within the range obtained by Ames et al. (1975) as well as reported by De Serres and Shelby (1979). Similarly, the results with the positive control substances confirmed the known reversion properties and specificity of the tester strains as well as the full activity of the metabolizing system.
Statistics:
The estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each
dosage level was calculated using the X2-test (Mohn and Ellenberger, 1977).
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the absence of S9 mix, bacteriotoxicity was observed at 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the absence of S9 mix, bacteriotoxicity was observed at 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the absence of S9 mix, bacteriotoxicity was observed at 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the absence of S9 mix, bacteriotoxicity was observed at 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the absence of S9 mix, bacteriotoxicity was observed at 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test compound an the plates was not observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the presence of S9-mix the substance was not bacteriotoxic.

Table 1: Number of revertants per plate (mean of three plates), Experiment 1 and 2, without metabolic activation

strain

TA 98

strain

TA 100

strain

TA 102

strain

TA 1535

strain

TA 1537

conc. [µg]

Exp1

Exp2

Exp1

Exp2

Exp1

Exp2

Exp1

Exp2

Exp1

Exp2

control

35

31

83

99

316

278

22

10

13

11

solvent control

32

31

88

90

265

257

24

10

11

11

5

33

 

87

 

183

26

 

8

 

15

34

20

79

79

191

165

27

14

9

13

50

39

30

88

78

249

214

29

14

11

13

150

35

28

82

85

239

236

12

11

9

9

500

28

26

86

84

118T

143T

20

11

7

9

1500

15T

47T

85T

 

7T

4T

Sodium azide

0.7 µg

 

 

528

499

 

 

841

471

 

 

2-nitrofluorene

2.5 µg

549

598

 

 

 

 

 

 

 

 

9-aminoacridine

50µg

 

 

 

 

 

 

 

 

115

180

MitomycinC

0.15 µg

 

 

 

 

807

881

 

 

 

 

T: Cytotoxicity

Table 2: Number of revertants per plate (mean of three plates), Experiment 1 and 2, with metabolic activation

strain

TA 98

strain

TA 100

strain

TA 102

strain

TA 1535

strain

TA 1537

conc. [µg]

Exp1

Exp2

Exp1

Exp2

Exp1

Exp2

Exp1

Exp2

Exp1

Exp2

control

27

26

89

90

289

320

12

11

17

10

solvent control

21

23

80

89

275

299

13

13

14

14

50

23

28

82

89

272

256

7

12

11

15

150

23

23

73

89

265

278

8

13

12

13

500

22

22

75

84

256

259

11

15

10

13

1500

20

23

74

82

235

273

15

15

9

15

5000

17

23

72

82

223

228

13

14

14

14

2-aminoanthracene

0.8 µg/plate

799

1018

917

753

 

 

 

 

 

 

2-aminoanthracene

0.9 µg/plate

 

 

 

 

538

 

308

91

 

 

2-aminoanthracene

1.0 µg/plate

 

 

 

 

 

592

 

 

 

 

2-aminoanthracene

1.7 µg/plate

 

 

 

 

 

 

 

 

114

203

T: Cytotoxicity

Conclusions:
The test substance was not mutagenic in the bacterial reverse mutation assay.
Executive summary:

The test substance was tested for its mutagenicity in the bacterial reverse mutation assay. Therefore, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 were used. The test was performed using the standard plate incorporation assay with and without S9 -mix (Aroclor 1254 induced). Two independet experiments were carried out using concentrations of 50 -5000 µg and 5 -1500 µg/plate. In the presence of S9-mix no bacteriotoxicity was observed. In the absence of S9-mix the test substance was bacteriotoxic towards the strain TA102 at 500 µg/plate and towards the strains TA1535, TA1537, TA98, and TA100 at 1500 µg/plate. As mutagenicity controls, sodium azide, 2 -nitrofluorene, 9 -aminoacridine and mitomycin C were used in the absence of S9 -mix and 2 -aminoanthracene was used in the in the presence of S9 mix. As a result of this assay, mutagenicity was not observed.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-06-02 to 2015-07-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: mammalian gene mutation assay (HPRT)
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (hprt) locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10% foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1%)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
mammalian microsomal fraction S9 mix
Test concentrations with justification for top dose:
Experiment I, exposure period 4 hours:
without S9 mix: 1.9, 3.8, 7.5, 15.1, 30.2, 60.3, 90.5, 120.7 µg/mL
with S9 mix: 60.3, 120.7, 241.3, 482.5, 965.0, 193.0 µg/mL

Experiment II, exposure period 24 hous:
without S9 mix: 7.5, 15, 30, 60, 120, 180, 240, 300 µg/mL
Exposure period 4 hours:
30, 60, 120, 240, 480, 960 µg/mL
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: with metabolic activation: DMBA
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 hours or 4 hours
- Expression time (cells in growth medium): 7 days

SELECTION AGENT: 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency



Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations of the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated 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. However, both, biological and statistical significance was considered together.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
4 h treatment: at 60.3 µg/mL (with and without S9 mix); after 24 h: at 241.3 µg/mL (without S9 mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: Phase separation occurred at 241.3 μg/mL and above after 4 and 24 hours treatment without metabolic activation and at 965.0 μg/mL and above with metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
A pre-test was performed in order to determine the concentration range of the mutagenicity experiments. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

COMPARISON WITH HISTORICAL CONTROL DATA:
A slight increase of mutant colonies per 106 cells exceeding the range of the historical solvent control data (1.6 - 45.7 mutant colonies/106 cells) was determined in both cultures of the first experiment at 60.3 μg/mL without metabolic activation (46.2 and 48.6 mutant colonies per 106 cells). However, the threshold of three times the corresponding solvent control was not exceeded and the effect was not reproduced in the second experiment without metabolic activation. Consequently, the isolated increases described above were judged as biologically irrelevant.

Conclusions:
The test item did not induce gene mutations at the HPRT locus in V79 cells.
Executive summary:

The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The maximum test item concentration of the pre-experiment (1930 μg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxicity (without metabolic activation) and phase separation (with metabolic activation) of the test item. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

The tested concentrations were:

Experiment I, exposure period 4 hours:

without S9 mix: 1.9, 3.8, 7.5, 15.1, 30.2, 60.3, 90.5, 120.7 µg/mL

with S9 mix: 60.3, 120.7, 241.3, 482.5, 965.0, 193.0 µg/mL

Experiment II, exposure period 24 hous:

without S9 mix: 7.5, 15, 30, 60, 120, 180, 240, 300 µg/mL

Exposure period 4 hours:

30, 60, 120, 240, 480, 960 µg/mL

Appropriate reference mutagens, used as positive controls (without metabolic activation: ethylmethane sulfonate, with metabolic activation: DMBA), induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Based on the available results the study was considered valid and the tets substance was shown to be not mutagenic in the HPRT assay.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015 to 2015-04-30
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes (HPBL)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: human donor
- Sex, age and number of blood donors:
preliminary test and initial assay: healthy non-smoking 27-year old adult female;
repeat assay: healthy non-smoking 24-year old adult female
- Separated lymphocytes were used

MEDIA USED
- Type and identity of media including CO2 concentration: complete medium (RPMI-1640; 15% fetal bovine serum, 2mM L-glutamine, 100 units penicillin, 100 µg/mL streptomycin) with 2% phytohemagglutinin; 37 °C; 5 +/- 1 % CO2
- Properly maintained: yes
Cytokinesis block (if used):
Cytochalasin B (cytoB) (6 µL/mL)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9 mix
Test concentrations with justification for top dose:
Test concentrations were chosen based on preliminary cytotoxicity test.
Initial test:
without S9, 4 h: 50, 100, 250, 300, 325, 350, 400 µg/mL
without S9, 24 h: 50, 100, 200, 250, 285, 300, 325, 350 µg/mL
with S9, 4 h: 100, 250, 500, 750, 1000 µg/mL

Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: recommeded by guideline and the test item was well soluble in the vehicle
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: vinblastine without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h and 24 h

SPINDLE INHIBITOR: cytochalasine B

STAIN: acridine orange

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: cells were collecetd by centrifugation and resuspended in fixative; fixed cells were applied to glass slides and air dried

NUMBER OF CELLS EVALUATED: min 2000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 2

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Micronuclei of a binucleated cell were recoreded it the meet the following criteria:
- the micronucleus should have the same staining characteristics as the main nucleus
- the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)
- the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity: % Cytostasis (cytotoxicity) = 100 - 100 ((CBPI t-1)/(CBPDI c-1))
where CBPI = 1x mononucleated cells + 2 x binucleated cells + 3 x multinucleated cells / total number of cells scored
Rationale for test conditions:
preliminary cytotoxicity test
Evaluation criteria:
The test substance would be considered positive if it induced a statistically significant and dose-dependent increase the frequency of MN-BN cells (p ≤ 0.05). If only one criterion was met (statistically significant OR dose-dependent increase), the result was considered equivocal. If neither criterion was met, the results were considered to be negative.
Statistics:
Statistical analysis of the percentage of micronucleated cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent micronucleated cells of each treatment group with that of the vehicle control. Due to negative results, Cochran-Armitage test was not required to measure dose-responsiveness.
Key result
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentration > 250 µg/mL without S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH in highest concentration was measured to be 7.5
- Effects of osmolality: none
- Evaporation from medium: no
- Precipitation: yes, at concentrations > 350 µg/mL without S9 mix and > 500 µg/mL with S9 mix
- Hemolysis was observed at concentrations of > 400 µg/mL without S9 mix

RANGE-FINDING/SCREENING STUDIES: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI
Conclusions:
Under the conditions of the assay the test item was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.
Executive summary:

The test substance was tested in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system.A preliminary toxicity was performed to establish the dose range for testing in the micronucleus test. The micronucleus assay was used to evaluate the aneugenic and clastogenic potential of the test substance. In the preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation.

Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells.In a solubility test the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

In the preliminary toxicity assay, the doses tested ranged from 0.192 to 1920 μg/mL (10 mM).Substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 576μg/mL in the non-activated 4 and 24-hour exposure groups.Substantial cytotoxicity was not observed at any dose level in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed in the treatment medium at dose levels ≥ 576 μg/mL in the non-activated 4 and 24-hour exposure groups and at 1920 μg/mL in the S9-activated 4-hour exposure group. Based on these findings, the doses chosen for the micronucleus assay ranged from 50 to 500 μg/mL for the non-activated 4 and 24-hour exposure groups, and from 100 to 1950 μg/mL for the S9-activated 4-hour exposure group.

In the initial micronucleus assay, substantial cytotoxicity was observed at dose levels ≥ 300 μg/mL in the non-activated 4-hour exposure group, and at dose levels ≥ 250 μg/mL in the non-activated 24-hour exposure group. Substantial cytotoxicity was not observed at any dose level in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed in the treatment medium at dose levels ≥ 750 μg/mL in the S9-activated 4-hour exposure group. However, due to technical reasons, the slides from the initial assay were not scorable. Therefore, the micronucleus assay was repeated at dose levels ranging from 50 to 400 μg/mL for the non-activated 4-hour exposure group, from 100 to 1000 μg/mL for the S9-activated 4-hour exposure group, and from 50 to 350 μg/mL for the non-activated 24-hour exposure group.

In the repeat assay, substantial cytotoxicity was observed at dose levels ≥ 250 μg/mL in the non-activated 4 and 24-hour exposure groups. Substantial cytotoxicity was not observed at any dose level in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed in the treatment medium at dose levels ≥ 750 μg/mL in the S9-activated 4-hour exposure group.

The highest dose analyzed under each treatment condition either exceeded the limit of solubility in treatment medium at the conclusion of the treatment period or produced 50 to 60% reduction in CBPI which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.

The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test). The results for the positive and negative controls indicate that all criteria for a valid assay were met.

Based on the findings of this study, the test item was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial reverse mutation assay

The test substance was tested for its mutagenicity in the bacterial reverse mutation assay (Symrise 2000485). Therefore, Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 were used. The test was performed using the standard plate incorporation assay with and without S9 -mix (Aroclor 1254 induced). Two independet experiments were carried out using concentrations of 50 -5000 µg and 5 -1500 µg/plate. In the presence of S9-mix no bacteriotoxicity was observed. In the absence of S9-mix the test substance was bacteriotoxic towards the strain TA102 at 500 µg/plate and towards the strains TA1535, TA1537, TA98, and TA100 at 1500 µg/plate. As mutagenicity controls, sodium azide, 2 -nitrofluorene, 9 -aminoacridine and mitomycin C were used in the absence of S9 -mix and 2 -aminoanthracene was used in the in the presence of S9 mix. As a result of this assay, mutagenicity was not observed.


HPRT assay

The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments. The cells were exposed to the test item for 4 hours in the first experiment with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The maximum test item concentration of the pre-experiment (1930μg/mL) was equal to a molar concentration of about 10 mM. The concentration range of the main experiments was limited by cytotoxicity (without metabolic activation) and phase separation (with metabolic activation) of the test item. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

The tested concentrations were:

Experiment I, exposure period 4 hours:

without S9 mix: 1.9, 3.8, 7.5, 15.1, 30.2, 60.3, 90.5, 120.7 µg/mL

with S9 mix: 60.3, 120.7, 241.3, 482.5, 965.0, 193.0 µg/mL

Experiment II, exposure period 24 hous:

without S9 mix: 7.5, 15, 30, 60, 120, 180, 240, 300 µg/mL

Exposure period 4 hours:

30, 60, 120, 240, 480, 960 µg/mL

Appropriate reference mutagens, used as positive controls (without metabolic activation: ethylmethane sulfonate, with metabolic activation: DMBA), induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Based on the available results the study was considered valid and the tets substance was shown to be not mutagenic in the HPRT assay.

In vitro micronuclei assay

The test substance was tested in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes (HPBL) in both the absence and presence of an Aroclor-induced S9 activation system.A preliminary toxicity was performed to establish the dose range for testing in the micronucleus test. The micronucleus assay was used to evaluate the aneugenic and clastogenic potential of the test substance. In the preliminary toxicity and the micronucleus assays, HPBL cells were treated for 4 and 24 hours in the non-activated test system and for 4 hours in the S9-activated test system. All cells were harvested 24 hours after treatment initiation.

Dimethyl sulfoxide (DMSO) was used as the vehicle based on the solubility of the test substance and compatibility with the target cells.In a solubility test the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

In the preliminary toxicity assay, the doses tested ranged from 0.192 to 1920 μg/mL (10 mM).Substantial cytotoxicity [≥ 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at dose levels ≥ 576μg/mL in the non-activated 4 and 24-hour exposure groups.Substantial cytotoxicity was not observed at any dose level in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed in the treatment medium at dose levels ≥ 576 μg/mL in the non-activated 4 and 24-hour exposure groups and at 1920 μg/mL in the S9-activated 4-hour exposure group. Based on these findings, the doses chosen for the micronucleus assay ranged from 50 to 500 μg/mL for the non-activated 4 and 24-hour exposure groups, and from 100 to 1950 μg/mL for the S9-activated 4-hour exposure group.

In the initial micronucleus assay, substantial cytotoxicity was observed at dose levels ≥ 300 μg/mL in the non-activated 4-hour exposure group, and at dose levels ≥ 250 μg/mL in the non-activated 24-hour exposure group. Substantial cytotoxicity was not observed at any dose level in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed in the treatment medium at dose levels ≥ 750 μg/mL in the S9-activated 4-hour exposure group. However, due to technical reasons, the slides from the initial assay were not scorable. Therefore, the micronucleus assay was repeated at dose levels ranging from 50 to 400 μg/mL for the non-activated 4-hour exposure group, from 100 to 1000 μg/mL for the S9-activated 4-hour exposure group, and from 50 to 350 μg/mL for the non-activated 24-hour exposure group.

In the repeat assay, substantial cytotoxicity was observed at dose levels ≥ 250 μg/mL in the non-activated 4 and 24-hour exposure groups. Substantial cytotoxicity was not observed at any dose level in the S9-activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed in the treatment medium at dose levels ≥ 750 μg/mL in the S9-activated 4-hour exposure group.

The highest dose analyzed under each treatment condition either exceeded the limit of solubility in treatment medium at the conclusion of the treatment period or produced 50 to 60% reduction in CBPI which met the dose limit as recommended by testing guidelines for this assay. A minimum of 1000 binucleated cells from each culture were examined and scored for the presence of micronuclei.

The percentage of cells with micronucleated binucleated cells in the test substance-treated groups was not statistically significantly increased relative to vehicle control at any dose level (p > 0.05, Fisher’s Exact test). The results for the positive and negative controls indicate that all criteria for a valid assay were met.

Based on the findings of this study, the test item was concluded to be negative for the induction of micronuclei in both non-activated and S9-activated test systems in the in vitro mammalian cell micronucleus test using human peripheral blood lymphocytes.


Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.