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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test, a chromosome aberration test in CHO cells and a mutagenicity assay in mammalien cells were performed according OECD guideline 471, 473 and 476 to evaluate the genotoxic potential of the test substance. The test item did neither induce mutations nor chromosome aberrations in vitro. Therefore, the substance is not considered to be genotoxic under the conditions of these tests.

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:
1992 - 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
In the Salmonella typhimurium strains (TA 1535, TA 100, TA 1537, TA 98) the amino acid histidine locus is the target gene, in which induced back mutations will transform the histidine auxotrophy (his-) to histidine prototrophy (his+). The Salmonella typhimurium histidine (his) reversion system measures his- => his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. The strain Escherichia coli (WP2 uvrA) is a tryptophan-auxotrophic strain (base-pair substitution).
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 mix (from liver homogenate of Aroclor 1254 treated rats)
Test concentrations with justification for top dose:
0, 312.5, 625, 1250, 2500, and 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent used: DMSO (50 mg/mL, suspension)
- Justification for choice of solvent/vehicle: DMSO was chosen because of its solubility properties and its relative nontoxicity
- On the day of the experiment (immediately before the experiment), the test item was dissolved in DMSO
Untreated negative controls:
other: = vehicle control
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: sodiuim azide, 4-NQO, 2-nitrofluorene (strains except TA 1537), 9-aminoacridine (TA 1537) - Without metabolic activation; 2-aminoanthracene (all strains), cyclophosphamide (all strains except TA 1537) - With metabolic activation
Remarks:
The solvent alone was used as the negative control.
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate method, no preincubation
Evaluation criteria:
The generally accepted conditions for the evaluation of the results are:
- mean colony counts of the control values of all strains are within the acceptable ranges
- results of the positive controls meet the criteria for a positive response

The test item is considered as mutagen in case of:
- reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration; Strains: S. typhimurium TA 98, TA 1535, TA 1537 and E. coli WP2 uvrA
- reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain S. typhimurium TA 100
- a concentration-related effect should be demonstrable.
Statistics:
A statistical analysis was not performed.
Key result
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
other: = vehicle control
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
other: = vehicle control
Positive controls validity:
valid
Additional information on results:
Concentration of test substance resulting in precipitation: approximately 600 - 5000.0 µg/plate.

Test item EC 411-380-6: Salmonella typhimurium/E.coli reverse mutation assay

Plate incorporation without S-9 mix: experiment I/II

Dose (µg/plate)

 TA 100

 TA 1535

 WP2 uvrA

 TA 98

 TA1537

 Negative control
(solvent alone)

131.7 / 139.3

12.0 / 10.3

17.0 / 14.7

21.7 / 13.0

6.0 / 7.0

 312.5

 112.7 / 129.3

 12.0 / 8.7

 17.0 / 16.7

 21.7 / 17.3

 4.3 / 

 625

 111.0 / 135.0

 11.3 / 9.0

 16 / 15.3

 21.7 / 12.7

 6.3 / 

 1250

 112.0 / 112.0

 11.3 / 7.0

 17 / 14.0

 18.0 / 12.3

 4.3 / 

 2500

 116.7 / 126.0

 8.3 / 7.3

 15 / 14.3

 19.0 / 17.0

 5.0 / 

 5000

 109.3 / 111.7

 10.3 / 7.3

 19 / 13.3

 21.7 / 14.0

 3.7 / 

Positive controls

 

 

 

 

 

- Sodum azide

 961.7 / 1092.3

 797.3 / 802.0

 

 

 

- 4-NQO

 

 

 758.0 / 935.7

 

 

2-nitrofluorene

 

 

 

 1209.7 / 1650.7

 

9-aminoacridine

 

 

 

 

 1436.3 / 1456.0

 

Plate incorporation with S-9 mix: experiment I/II

Dose (µg/plate)

 TA 100

 TA 1535

 WP2 uvrA

 TA 98

 TA1537

 Negative control
(solvent alone)

140.3 / 140.0

12.0 / 10.3

18.3 / 19.0

46.7 / 30.0

8.7 / 6.7

 312.5

 131.7/ 133.7

 12.0 / 9.3

 22.3 / 15.7

 37.3 / 27.3

 7.7 / 8.7

 625

 118.7 / 129.7

 11.7 / 14.0

 22.0 / 14.3

 36.0 / 28.3

 7.3 / 7.0

 1250

 120.0 / 112.0

 10.7 / 12.7

 21.3 / 13.3

 38.0 / 22.3

 7.3 / 6.7

 2500

 118.7 / 124.7

 13.0 / 10.3

 16.7 / 14.0

 43.0 / 24.7

 8.0 / 7.0

 5000

 129.0 / 96.7

 10.7 / 9.7

 16.7 / 20.3

 38.3 / 30.0

 5.0 / 4.3

Positive control

 

 

 

 

 

- 2-Aminoanthracene

 1650.7 / 1432.3

 

 1084.3 / 733.3

 1917.7 / 1785.7

 249.3 / 185.7

- Cyclophosphamide

 

 374.0 / 362.3

 

 

 

Revertants/plate (I / II): mean from three plates     

Conclusions:
Interpretation of results: negative
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium and Escherichia coli strain WP2 uvrA were exposed to the test item (as described in section 1.2) dissolved in DMSO at concentrations of 312.5, 625, 1250, and 5000.0 µg/plate in the presence and absence of mammalian metabolic activation without pre-incubation. Concurrent positive and solvent controls were included. All concentrations were tested in triplicates. Concentrations of approximately 600 - 5000.0 µg/plate resulted in precipitation of the test item; no cytoxicity was noted up to the limit concentration. The positive controls induced the appropriate responses. No evidence of a mutagenic potential associated with the test substance was observed. This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
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 OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
, MITI (March 31, 1987)
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test
Target gene:
No target gene
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Culture conditions:
CHO cell line CCL 61 was maintained in culture medium consisting of Nutrient Mixture F-12 supplemented with 10% fetal calf serum + Penicillin/Streptomycin 100 units/mL / 100 µg/mL (Gibed AG, Basle, Switzerland) in 75 cm² tissue-culture (plastic) flasks. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2. The cells were passaged twice weekly. The cell cultures were periodically checked for mycoplasma contamination. The duration of a normal cell cycle determined was 12 - 13 hours.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat-liver homogenate (S9).
Test concentrations with justification for top dose:
without S9 mix:
18 h; 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 µg/mL (experiment 1)
42 h; 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 µg/mL (experiment 3)

with S9 mix:
3 h; 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 µg/mL (experiment 2; recovery 15 h)
3 h; 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 µg/mL (experiment 4; recovery 39 h)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: As the test item was insoluble in all common vehicles applicable in the test system, it was tested as a suspension in acetone (stock solution: 50 mg test item/mL acetone).
Negative solvent / vehicle controls:
yes
Remarks:
Negative control, supplemented with the respective volume of the vehicle
Positive controls:
yes
Positive control substance:
other: without S9: Mitomycin C (0.2 µg/mL); with S9: Cyclophosphamide (20 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation time before treatment: 29 h
- Treatment in the absence of metabolic activation: throughout the whole period
- Treatment in the presence of metabolic activation three hours, prolonged exposure would result in cytotoxicity
- To ensure analysis of first post treatment mitoses a major sampling time of 1.5 times cell cycle was selected (about 18 hours). In case of a delayed cell cycle, an additional sampling time was chosen, 24 hours, after the first one.
- Original study, first experiment: 18 h without metabolic activation
- Original study, second experiment: 3 h with metabolic activation
- Confirmatory study, first experiment: 18 h without metabolic activation
- Confirmatory study, second experiment: 3 h with metabolic activation
- Confirmatory studv. third experiment: 42 h without metabolic activation
- Confirmatory study, fourth experiment: 3 h with metabolic activation
- After treatment with metabolic activation cells were washed and incubated in new complete culture medium for 15 hours.
- Then a spindle inhibitor was added to the cultures. After 2.0 h the cells were treated on the slides with hypotonic solution. Afterwards the cells were fixed and finally stained.

SPINDLE INHIBITOR (cytogenetic assays): Colcemide (0.4 µg/mL culture)

STAIN (for cytogenetic assays): DAPI (4',6-diamino-2-phenylindole), Serva, Germany. Fluorescence of DAPI stained DNA was measured with a flow cytophotometer. A substantial shift in the DNA distribution pattern of cell cultures in comparison with the pattern of the vehicle control would indicate a disturbance of the cell cycle induced by the test item.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 metaphases per replicate culture

DETERMINATION OF CYTOTOXICITY
- integral part of the mutagenicity test
Evaluation criteria:
The test item is generally considered to be active in the Chinese hamster cells if:
- The percentage of metaphases containing specific aberrations in a treatment group is higher than 6.0 (based on historical negative control range) and differs statistically significant from the respective value of the negative control.
- A concentration-related response should be demonstrable

The test item is generally considered to be inactive in the Chinese hamster cells if:
- The percentage of metaphases containing specific aberrations in all treatment groups is less than or equal to 6.0 (based on historical negative control range) and does not differ statistically significant from the respective value of the negative control.
Statistics:
The evaluated numbers of specific aberrations were subjected to statistical analysis. In the preliminary tests the data were assessed for flask effects (dependence of cells within each culture) using a chi-square test. Accordingly a chi-square test for trend was performed modelling all cells in a given experiment as independent. That is, the individual cell is taken as the experimental unit. Consequently the power of the test is substantially increased, resulting in a rather save judgement of the observed effects. The tests were performed based upon the presence of any specific aberration.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the presence of S9 mix some toxic effects were indicated by reduced Mitotic Index Values
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Clastogenic activity was assessed at concentrations of 125, 250, 500 µg/mL.
Conclusions:
Interpretation of results: negative
Executive summary:

In a mammalian cell cytogenetics assay to evaluate chromosomal aberration, Chinese hamster ovary (CHO) cell cultures were exposed to the test item (as described in section 1.2) dissolved in acetone at concentrations of 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 µg/mL with or without metabolic activation. Clastogenic activity was assessed at concentrations of 125, 250, and 500 µg/mL. Preparation of chromosomes of assays without metabolic activation was done immediately after 18 h and 48 h exposure period. Preparation of chromosomes of assays with metabolic activation was done after 3 h exposure period followed by a recovery period. In the presence of S9 mix some toxic effects were indicated at higher test item concentrations by reduced Mitotic Index Values. Each concentration was tested in duplicates. 200 metaphases were counted per concentration. Positive controls induced the appropriate response. No evidence of a clastogenic potential was observed. This study satisfies the requirement for Test Guideline „In Vitro Mammalian Chromosome Aberration Test“ OECD 473 for in vitro cytogenetic mutagenicity data in general.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
other: mammalian cell gene mutation assay
Target gene:
HGPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S-9 microsomal activation system
Test concentrations with justification for top dose:
0, 18.52, 55.56, 166.67 and 500 microg/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
without metabolic activation Migrated to IUCLID6: DMN, 1.0 microliter/ml
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
with metabolic activation Migrated to IUCLID6: 0.3 microliter/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: with metabolic activation for 5 hours and in the experiment without metabolic activation for 21 hours

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: two

NUMBER OF CELLS EVALUATED: all colonies were counted

DETERMINATION OF CYTOTOXICITY
Cytotoxicity test
In the part with metabolic activation, at the highest concentration of 500 microg/ml an acute growth inhibiting effect of 55.15% could be seen. Withoutmetabolic activation treatment was growth inhibiting by 39.42% at the highest concentration of 500 microg/ml. Accordingly, 500 microg/ml was chosen as the highest concentration for the first mutagenicity assay with and without metabolic activation. Precipitates of the test compound were visible in the cultures down to the concentration of 62.5 ug/ml.

OTHER
The cells have a stable karyotype with a modal chromosome number of 22±1. All stock cells were checked for mycoplasma contamination, using the Hoechst- Dye staining method or the 6-MPDR method, before being frozen.
Evaluation criteria:
All mutant frequencies are normalized to a virtual cloning efficiency of 100% at the end of the expression
period. If the cloning efficiency of the viability cultures is lower than 15%, the corresponding
mutant frequency is usually not calculated, owing to the high statistical insignificance of the
result. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant
frequencies of the treated cultures with the mean mutant frequencies of the solvent control cultures,
will be calculated.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitates of the test compound were visible in the cultures down to the concentration of 62.5 microg/ml.

RANGE-FINDING/SCREENING STUDIES:

COMPARISON WITH HISTORICAL CONTROL DATA: Historical control data from gene mutation tests with Chinese Hamster Cells V79

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the part with metabolic activation, at the highest concentration of 500 microg/ml an acute growth inhibiting effect of 55.15% could be seen. Without metabolic activation treatment with the test item was growth inhibiting by 39.42% at the highest concentration of 500 microg/ml. Accordingly, 500 microg/ml was chosen as the highest concentration for the first mutagenicity assay with and without metabolic activation.
Remarks on result:
other:
Conclusions:
Interpretation of results: negative

Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that the test item and its metabolites did not show any mutagenic activity in this forward mutation system.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Procedure and observations

An Ames test, a test of chromosome aberration in V79 cells and an HPRT assay were performed according OECD guideline 471 (Ciba 1992f), 473 (Ciba1993b) and 476 (Ciba 1994), respectively, to evaluate genotoxicity of the test substance in vitro. All three studies were performed under GLP.

The Ames test was conducted to investigate the potential of the test article to induce gene mutations according to the plate incorporation test (experiment I and II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in addition the Escherichia coli strain WP2 uvrA. The assay was performed with and without induced rat liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article, dissolved in DMSO, was tested at the following concentrations: 0, 312.5, 625, 1250, 2500, and 5000 µg/plate.

The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without metabolic activation in all independent experiments. Concentrations of approximately 600 - 5000.0 µg/plate resulted in precipitation of the test item; no cytoxicity was noted up to the limit concentration. No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with the test substance at any dose level, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no relevant tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

In a mammalian cell cytogenetic assay to evaluate chromosomal aberration, Chinese hamster ovary (CHO) cell cultures were exposed to the test item dissolved in acetone at concentrations of 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500 µg/ml with or without metabolic activation, clastogenicity was evaluated at concentrations of 125, 250, and 500 µg/mL. Preparation of chromosomes of assays without metabolic activation was done immediately after 18 h and 48 h exposure period. Preparation of chromosomes of assays with metabolic activation was done after 3 h exposure period followed by a recovery period. In the presence of S9 mix some toxic effects were indicated at higher test item concentrations by reduced Mitotic Index Values. Each concentration was tested in duplicates. 200 metaphases were counted per concentration. Positive controls induced the appropriate response. No evidence of a clastogenic potential was observed.

The mutagenicity assay in mammalien cells (V79) was performed at the following concentrations: 18.52, 55.56, 166.67 and 500 microg/ml in four independent experiments (two with and without metabolic activation). No growth inhibition was found both after treatment and expression. N-Nitrosodimethylamine (DMN, 1.0 microliter/ml, without S9 -mix) and ethylmethanesulfonate (EMS, 0.3 microliter/ml, with metabolic activation) were used as positive controls. In all experiments comparison of the number of mutant colonies in the controls and in the cultures treated with the various concentrations of the test substance revealed no relevant increase of the mutant frequencies as determined by the screening with 6-Thioguanine (6 -TG). Precipitates of the test substance in the culture medium were visible at the two higher concentrations.

Discussion

Treatment of different bacteria strains or mammalian cells with the test substance in presence and absence of S9-mix did not induce increases in revertant colonies or mutant frequencies, respectively. Incubation of CHO or V79 cells with the test substance in presence and absence of S9-mix did not induce chromosome aberrations, polyploidy metaphases or an increase in mutant frequencies, respectively. Therefore, the substance is not considered to be genotoxic under the conditions of these tests.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for genotoxicity under Directive 67/548/EEC, as amended for the 30th time in Directive 2008/58/EC.

 

 

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008, as amended for the fifth time in Directive EC 944/2013.