Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An in vitro gene mutation study on bacteria was conducted on the registered substance concluded it was not mutagenic under the conditions of the test, with and without metabolic activation.


In vitro genotoxicity and mutation studies conducted on mammalian cells using a suitable analogue concluded that it was not mutagenic under the conditions of the test, with and without metabolic activation. These studies are deemed as acceptable to conclude on the properties of the registered substance.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: before and after treatment RPMI 1640 with 2 mM glutamine, 100 units/mL penicillin, 100 µg/mL streptomycin and 10% horse serum (referred to as R10); for cloning for survival and mutation cells were grown in the same base medium containing 20% horse serum instead and additionally 200 µg/mL sodium pyruvate (referred to as R20). In the range finder and during treatment the same base medium with 5% horse serum was used (referred to as R5).
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
beta-naphthoflavone and sodium phenobarbitone-induced rat liver S-9
Test concentrations with justification for top dose:
10, 25, 50, 75 and 100 µg/mL ± S-9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: positive control chemicals in DMSO, test substance in ethanol (2% v/v final concentration in culture)
- Justification for choice of solvent/vehicle: low solubility of substances in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S-9 Migrated to IUCLID6: 750 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S-9 Migrated to IUCLID6: 25µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): 10-12 days (5% Co2, 37 °C)
- Fixation time (start of exposure up to fixation or harvest of cells): recording of colony formation on days 13-15

SELECTION AGENT (mutation assays): TFT (trifluorothymidine)

NUMBER OF REPLICATIONS: 384 wells per concentration analysed; 2 independent experiments performed

NUMBER OF CELLS EVALUATED: 2000 cells/well (10e4 cells/mL) initially plated in 4 96-well plates for assessment of mutations at the TK locus

DETERMINATION OF CYTOTOXICITY
- Method: plating efficiency: cultures were diluted to 8 cells/mL in R20, and 0.2mL aliquots of this suspension were dispensed into 2 96-well microtitre plates for each dose point (average of 1.6 cells/well).
Evaluation criteria:
Positive: A dose response curve with at least one point showing a statistically significant increase in mutation frequency (mutations per 10e6 survivors) compared to the solvent control, which is reproducible in both experiments.

Negative: Neither a dose response curve nor any statistically significant increases in mutation frequency are observed.
Statistics:
The distribution of colony forming units amongst the wells of a microtitre plate follows the Poisson Distribution, so that the plating efficiency (PE) is calculated using the zero form of the Poisson Distribution:
P(0) = number of empty wells/total wells plated

PE = -ln P(0)/n
(n = number of cells plated per well)

The mutation frequency (MF) per survivor is given by:
MF = PE(s)/PE(m) = Plating efficiency in selective medium/Plating efficiency in non selective medium

All individual mutation assessment points are compared to the controls, using the comparison of multiple treatments with control described in "Statistical Evaluation of Mutagenicity Test data" (Ed. D.J. Kirkland published by Cambridge University Press 1989).
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: test substance dissolved in ethanol due to very low water solubility
- Other: There was a statistically significant difference in the negative (solvent) control's mutation frequency compared to the untreated controls in the abence of S-9 only in the first experiment. No comparable effect was observed in the second experiment.

RANGE-FINDING/SCREENING STUDIES:
Logarithmically growing L5178Y cells were suspended in R5 at 5x10e5 cells/mL and treated with the test substance for 3 hours at 37 °C in both the absence and presence of S-9 mix. At the end of the treatment period, the cells were washed with phosphate buffered saline to remove the test substance, resuspended and counted. Aliquots of each culture were diluted to 8 cells/mL and plated in R20 to measure survival. From the resuls of the range finder, doses of 10, 25, 50, 75 and 100 µg/mL in the absence and presence of metabolic activation were chosen for the main experiments.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
the limiting factor for dose administration was the solubility limit for the test article. There was no toxicity demonstrated in the range finder.

Mutant frequencies mouse lymphoma assay:

Dose (µg/mL)

-S9

+S9

Mutation frequency

t

Mutation frequency

t

Summary of Mutant frequencies for Experiment 1

25

1.05e-3

10.10*

4.74e-4

5,97*

50

6.83e-4

2.93

4.19e-4

3.39

75

3.04e-4

2.15

1.87e-4

2.74

100

5.77e-4

1.18

1.67e-4

4.49

Solvent control

4.42e-4

16.1*(vs. medium)

2.83e-4

0.6 (vs. medium)

Medium

2.41e-4

-

3.37e-4

-

EMS

1.09e-3

36.4*

-

-

B(a)P

-

-

2.24e-3

203.8*

Summary of Mutant Frequencies for Experiment 2

25

2.52e-4

1.24

1.53e-4

0.92

50

2.34e-4

1.84

1.40e-4

1.78

75

1.79e-4

5.86

1.47e-4

1.29

100

3.45e-4

0.03

1.39e-4

1.89

Solvent control

3.32e-4

1.3 (vs. medium)

1.94e-4

0.0 (vs. medium)

Medium

2.78e-4

-

1.95e-4

-

EMS

1.21e-3

80.1*

-

-

B(a)P

-

-

4.22e-4

29.0

* = p<0.05

Compared to the relevant negative controls the cells treated with the positive control chemicals had significantly increased mutation frequencies (p<0.05).

In experiment 1 there was a statistically signifcant difference in the negative (solvent) control's mutation frequency compared to the untreated controls in the absence of metabolic activation only. There were no statistically significant differences in the mutation frequencies of negative (solvent) controls compared to the untreated cultures in experiment 2. As that observation was not reproducible in the second experiment it was not considered to be of biological significance.

In experiment 1 the only statistically significant increase in mutation frequency was observed after dosing with the test substance at 25 µg/mL with and without metabolic activation. There was no evidence of a positive dose response. In experiment 2 there were no statistically significant increases in mutation frequency observed after dosing with the test substance.

Conclusion:

The test substance did not show mutagenic activity under the conditions of this test.

Conclusions:
Interpretation of results (migrated information):
negative

The test material is not mutagenic to mouse Lymphoma cells.
Executive summary:

In an OECD 476 study, conducted according to GLP, the structurally similar read across substance is non-mutagenic to mouse lymphoma.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: primary cells from peripheral human blood
Metabolic activation:
with and without
Metabolic activation system:
beta-naphthoflavone and sodium phenobarbitone-induced rat liver S9
Test concentrations with justification for top dose:
Exp 1: -S9: 0.5, 2.5, 5, 25, 50 µg/mL; +S9: 10, 50, 100 µg/mL
Exp 2: -S9: 5, 25, 50 µg/mL; +S9: 10, 50, 100 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (2% v/v with S9 mix and 1% v/v without S9 mix)
- Justification for choice of solvent/vehicle: low solubility of test material in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S-9 Migrated to IUCLID6: final conc. 0.3 µg/mL
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S-9 Migrated to IUCLID6: final conc. 15 µg/mL and 20 µg/mL, respectively
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Experiment 1: 3 hours with S9, 20 hours (1.5 cell cycles) without S9; Experiment 2, Set 1+2: 3 hours with S9, 20 hours without S9
- Expression time (cells in growth medium): Experiment 1: 20 hours (1.5 cycle times, cycle length 12.5 hours) with/without S9; Experiment 2, set 1: as experiment 1; Experiment 2, set 2: 44 h with/without S9
- Fixation time (start of exposure up to fixation or harvest of cells): Experiment 1: 23 hours with S9, 20 hours without S9; Experiment 2, set 1: as experiment 1; Experiment 2, set 2: 47 hours with S9, 44 hours without S9

SPINDLE INHIBITOR (cytogenetic assays): colcemid (0.2 µg/mL), 2 hours before harvest
STAIN (for cytogenetic assays): 5% Gurrs Giemsa R66, pH 6.8

NUMBER OF REPLICATIONS: duplicates (2 cultures from different blood donors)

NUMBER OF CELLS EVALUATED: 200 (100 from each donor), solvent controls 400

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (minimum of 1000 cells)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes, cells with more than 48 chromosomes were recorded as numerical aberrations.
- Determination of endoreplication: yes

OTHER: only well spread metaphases with a chromosome count of 44-48 were scored for chromosome damage.
Evaluation criteria:
Positive: A dose response with at least one point having statistically more aberrations (p<0.05) than the solvent control is required. As the complex interaction between cell cycle and induced damage can give reduced damage scores at higher dose levels, the dose response curve will not always be a strict increase in damage with dose.

Negative: Neither a dose response nor any statistically significant increases in aberrations were observed.
Statistics:
Analysis of both total aberrations and percent aberrant cells by Fisher's exact test, as recommended by UKEMS ("Statistical Analysis of Mutagenicity Test Data" (1989) ed. D.J. Kirkland, Cambridge University Press).
Species / strain:
lymphocytes: primary cells from peripheral human blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
tested concentrations limited by solubility in the maximum applicable solvent concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects on pH were observed: pH 7.3 in the absence of S9
- Effects of osmolality: Osmolality in the absence of S9: medium 272.270 mOsm, 2% ethanol 612.619 mOsm, 100 µg/mL test substance concentration 671.668 mOsm
- Water solubility: limited by maximum applicable solvent concentration of 2% (v/v) ethanol: 50 and 100 µg/mL test substance without and with S9, respectively

RANGE-FINDING/SCREENING STUDIES:
In order to avoid toxicity to the cultures the maximum concentration of ethanol added was 2% (v/v). This limited maximum treatment concentration to 100 µg/mL. Osmolality and pH measurements were taken of medium control and preparations of 2% ethanol and 100 µg/mL test material in treatment medium without S9. The cells were treated either with medium only, medium containing 2% (v/v) ethanol as solvent control, or a preparation of the test material in ethanol added to medium at 2% (v/v). The concentrations of test material were 1, 5, 10, 50 and 100 µg/mL. Cultures were also included for treatment with 1% ethanol as an extra control.

Results demonstrated effects of 2% ethanol on the cultures (reduction of mitotic index) in the absence of S9; therefore, for the actual experiments 1% (v/v) ethanol was chosen as solvent concentration in the cultures without S9. This limits the maximum attainable test substance concentration in the absence of S9 to 50 µg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA:
The control data was well comparable with that of the historic controls.

Chromosomal Aberration Experiment 1:

Dose (µg/mL)

Total cells

Aberrant cells

Mitotic Index

Gaps

% cells with aberrations

with gaps

w/o gaps

Without metabolic activation, exposure period 20h, fixation time 20h (1% v/v ethanol)

5

200

5

6.75

0

2.5

2.5

25

200

5

7.05

3

2.5

1

50

200

6

10.60

2

3

2

solvent

400

7

6.23

4

1.8(*)

1.3

untreated

200

1

11.55

0

0.5

0.5

MMC

159

61

4.15

6

38.4(<***)

37.1(<***)

With metabolic activation, exposure period 3h, fixation time 20h (2% v/v ethanol)

10

200

9

6.20

3

4.5(*)

3(**)

50

200

3

4.85

1

1.5

1

100

200

6

5.85

1

3

2.5(*)

solvent

400

9

6.68

7

2.3

0.5

untreated

200

6

5.70

5

3

0.5

CPA

133

51

1.30

20

38.3(<***)

30.8(<***)

* = p<0.05

** = p<0.01

*** = p<0.001

<*** = p<0.00005

 

Chromosomal Aberration Experiment 2:

Dose (µg/mL)

Total cells

Aberrant cells

Mitotic Index

Gaps

% cells with aberrations

with gaps

w/o gaps

Without metabolic activation, exposure period 20h, fixation time 20h (1% v/v ethanol)

5

200

2

10.90

1

1

0.5

25

200

3

9.65

1

1.5

1

50

200

5

11.45

1

2.5

2

solvent

400

13

9.15

4

3.3

2.3

untreated

200

4

10.40

1

2

1.5

MMC

200

62

2.95

7

31(<***)

28.5(<***)

With metabolic activation, exposure period 3h, fixation time 20h (2% v/v ethanol)

10

200

2

6.50

2

1

0.5

50

200

4

6.20

1

2

1.5

100

200

3

8.30

1

1.5

1

solvent

400

5

6.68

2

1.3

0.8

untreated

200

2

11.15

1

1

0.5

CPA

200

33

1.70

12

16.5(<***)

13(<***)

Without metabolic activation 2nd harvest, exposure period 20h, fixation time 44h (1% v/v ethanol)

50

200

1

3.85

0

0.5

0.5

solvent

400

12

5.60

7

3

1.3

untreated

200

7

7.80

4

3.5

2

With metabolic activation 2nd harvest, exposure period 3h, fixation time 44h (2% v/v ethanol)

100

200

5

3.65

3

2.5

1

solvent

400

3

6.08

1

0.8

0.5

untreated

200

3

8.70

3

1.5

0

<*** = p<0.00005

All experimental points were compared statistically by the Fisher's exact test with the relevant negative controls (solvent). Solvent control values were compared statistically by the fisher's exact test with the relevant medium controls (untreated).

The positive control chemicals have caused statistically significant increases in the number of aberrations scored in both experiments. This demonstrates that the cells were sensitive to the effects of a known clastogen.

Treatment with the test substance showed some statistically significant increases in aberrations. These were not dose related or consistent between donors or experiments and are therefore not thought to demonstrate clastogenicity.

Conclusion:

The test substance has not demonstrated a consistent clastogenic effect under the conditions of this test.

 

Conclusions:
The test materail is not clastogenic to Human Lymphocytes.
Executive summary:

In a read across study a similar substance is non-clastogenic to human lymphocytes.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
April to June 2013
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
Target gene:
histidine or
tryptophan locus
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
S-9
Test concentrations with justification for top dose:
Experiment 1: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate.
Experiment 2: 50, 150, 500, 1500 and 5000 µg/plate.
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Acetone
Justification for choice of solvent/vehicle: The test material was initially noted to be insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL and acetone at 100 mg/mL but was fully soluble in tetrahydrofuran at 200 mg/mL. However, the test material in tetrahydrofuran could not be adequately dosed because the formulation immediately solidified during the pipetting stage. Therefore, the most suitable suspension was selected. The test material formed the best doseable suspension in acetone, therefore, this solvent was selected as the vehicle.
Untreated negative controls:
yes
Remarks:
ENNG: 2µg/plate for WP2uvrA, 3µg/plate for TA100, 5µg/plate for TA1535; 9AA: 80µg/plate for TA1537; 4NQO: 0.2µg/plate for TA98.
Negative solvent / vehicle controls:
yes
Remarks:
Acetone
True negative controls:
not specified
Positive controls:
yes
Remarks:
2AA: 1µg/plate for TA100, 2µg/plate for TA1535 and TA1537, 10µg/plate for WP2uvrA; BP: 5µg/plate for TA98.
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Acetone is toxic to the bacterial cells at 100µl after employing the pre-incubation modification, therefore all of the formulation for Experiment 2 were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50µL) aliquots.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The test material was considered to be non-mutagenic under the conditions of the study.
Executive summary:

In an OECD 471 study, conducted according to GLP, the test material is non-mutagenic (negative) to Salmonella typhimurium and Escherichia coli bacterial strains.

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

Additional information

Justification for classification or non-classification

In vitro testing conducted on the registered substance returned negative results, with and without metabolic activation. According to Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures (CLP), the registered substance does not meet the criteria for classification.