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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was not mutagenic in an Ames test (Toxikon, 2006), not clastogenic in a chromosome aberration test (Toxikon, 2006) and not mutagenic in an HPRT test (Harlan, 2013)

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 02, 2012 - March 06, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Remarks:
Harlan Cytotest Cell Research GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT (hypoxanthine-guanine phosphoribosyl transferase)
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 checked for spontaneous mutant frequency: yes
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Experiment I:
4 hours, without S9 mix: 13.0, 26.0, 52.0, 104.0, 208.0, (416.0) µg/ml
4 hours, with S9 mix: 13.0, 26.0, 52.0, 104.0, 208.0, (416.0) µg/ml
Experiment II:
24 hours, without S9 mix: 0.8, 1.6, 3.3, 6.5, 13.0, (26.0), (52.0), (104.0) µg/ml
4 hours, with S9 mix: (13.0), 26.0, 52.0, 104.0, 208.0, 416.0 µg/ml
numbers in parantheses: these cultures were discontinued.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: THF (tetrahydrofuran)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
without S9: EMS (0.15 mg/ml); with S9: DMBA (1.1 µg/ml)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (with and without S9), 24h (without S9)
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 18 - 20 days

SELECTION AGENT (mutation assays): 11 μg/mL 6-thioguanine
STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: two independent cultures were used

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency, cell density
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 fre¬quency at least at one of the concen-trations in 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 number of mutant colonies obtained for the groups treated 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 relevance and statistical significance was considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At 13.0 µg/ml and higher without S9 mix and 24 hour treatment.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Phase separation, visible to the unaided eye, was noted at 52.0 µg/mL and above in experiment I with and without metabolic activation. In experiment II phase separation occurred at the maximum concentration of 416.0 µg/mL in the presence of metabolic activation.

RANGE-FINDING/SCREENING STUDIES:
No relevant toxic effect occurred up to the maximum concentration with and without metabolic activation following 4 and 24 hours treatment. The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Phase separation occurred at 207.5 µg/mL and above in the presence and absence of metabolic activation following 4 and 24 hours treatment. In the pre-experiment there was no relevant shift of osmolarity and pH-value of the medium even at the maximum concentration of the test item. Based on the results of the pre-experiment, the individual concentrations of the main experiments were selected.

COMPARISON WITH HISTORICAL CONTROL DATA:
In the first experiment, culture I, the mutation frequency exceeded the historical range of solvent controls (3.4 - 36.6 mutant colonies/10^6 cells) at 13.0 and 26.0 μg/mL with metabolic activation (38.0 and 48.1 mutant colonies/10^6 cells). However, the induction factor did not exceed the threshold of three times the corresponding solvent control and statistical analysis showed that there was no dose dependent increase. Therefore, the increases were judged as biologically irrelevant.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures solely occurred in the second experiment at the highest analysable concentration of 13.0 µg/mL without metabolic activation (24 hours treatment). Exceedingly severe cytotoxic effects precluded analysis of the data at the next higher concentration of 26.0 µg/mL.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Summary of Results

concentration (µg/ml) PS S9 Mix relative cloning efficiency I (%) relative cell density (%) relative cloning efficiency II (%) mutant colonies / 106cells induction factor relative cloning efficiency I (%) relative cell density (%) relative cloning efficiency II (%) mutant colonies / 106cells induction factor
Experiment I / 4h treatment culture I culture II
solvent control - 100.0 100.0 100.0 32.0 1.0 100.0 100.0 100.0 35.7 1.0
positive control (EMS) 150.0 - 103.2 118.1 98.9 96.5 3.0 163.0 104.2 86.9 123.0 3.4
test item 13.0 - 27.1 39.5 99.3 18.4 0.6 49.4 39.1 99.6 30.9 0.9
test item 26.0 - 52.9 44.7 97.8 1.2 0.0 66.6 54.9 91.6 16.4 0.5
test item 52.0 PS - 58.5 53.0 111.5 12.3 0.4 63.7 51.4 91.8 15.7 0.4
test item 104.0 PS - 61.8 66.1 104.4 24.6 0.8 73.9 77.0 88.2 18.4 0.5
test item 208.0 PS - 82.0 101.1 109.6 11.5 0.4 90.7 78.3 92.6 17.4 0.5
test item 416.0 PS - 87.9 culture was not continued# 94.8 culture was not continued#
solvent control + 100.0 100.0 100.0 32.1 1.0 100.0 100.0 100.0 36.5 1.0
positive control (DMBA) 1.1 + 94.0 104.7 87.9 411.9 12.8 92.6 95.4 93.8 231.6 6.3
test item 13.0 + 103.7 103.5 101.2 38.0 1.2 95.4 89.1 115.1 14.4 0.4
test item 26.0 + 96.9 109.8 84.2 48.1 1.5 94.2 99.8 97.5 20.1 0.6
test item 52.0 PS + 95.5 101.0 93.4 16.9 0.5 94.5 114.1 115.5 36.0 1.0
test item 104.0 PS + 96.0 106.7 85.2 17.8 0.6 86.5 91.0 110.4 21.2 0.6
test item 208.0 PS + 93.6 111.3 87.9 27.6 0.9 88.1 113.7 110.9 33.0 0.9
test item 416.0 PS + 96.7 culture was not continued# 89.9 culture was not continued#
Experiment II / 24h treatment culture I culture II
solvent control - 100.0 100.0 100.0 18.5 1.0 100.0 100.0 100.0 5.5 1.0
positive control (EMS) 150.0 - 99.2 121.4 91.4 400.9 21.7 95.2 93.0 64.5 370.1 67.9
test item 0.8 - 101.1 115.2 97.0 13.5 0.7 101.0 112.7 94.0 6.4 1.2
test item 1.6 - 104.9 102.2 85.8 16.6 0.9 101.1 90.6 88.9 14.2 2.6
test item 3.3 - 94.7 102.2 94.5 17.2 0.9 98.9 98.8 95.5 10.4 1.9
test item 6.5 - 89.8 118.1 89.8 10.4 0.6 68.6 89.7 88.9 9.9 1.8
test item 13.0 - 41.5 43.9 96.7 11.8 0.6 40.7 40.2 89.5 4.3 0.8
test item 26.0 - 0.0 8.6 culture was not continued## 0.0 6.2 culture was not continued##
test item 52.0 PS - 0.0 3.5 culture was not continued## 0.0 3.5 culture was not continued##
test item 104.0 PS - 0.0 5.8 culture was not continued## 0.0 5.3 culture was not continued##
Experiment II / 4h treatment culture I culture II
solvent control + 100.0 100.0 100.0 8.9 1.0 100.0 100.0 100.0 10.8 1.0
positive control (DMBA) 1.1 + 78.0 63.7 68.9 724.0 81.2 71.4 97.4 67.9 1071.7 99.2
test item 13.0 + 103.6 culture was not continued### 98.8 culture was not continued###
test item 26.0 + 110.1 79.4 87.4 12.2 1.4 107.6 113.2 95.9 15.4 1.4
test item 52.0 + 92.6 77.7 98.4 9.0 1.0 97.3 113.6 90.1 7.3 0.7
test item 104.0 + 92.1 72.6 102.7 4.8 0.5 103.7 109.1 101.2 10.9 1.0
test item 208.0 + 96.3 81.6 98.0 9.5 1.1 108.2 118.9 95.9 13.8 1.3
test item 416.0 PS + 95.8 81.1 84.7 11.8 1.3 103.6 113.2 90.1 9.6 0.9

PS phase separation visible at the end of treatment

# culture was not continued to avoid analysis of too many concentrations displaying phase separation

## culture was not continued due to severe cytotoxic effects

### culture was not continued as a minimum of only four analysable concentrations is required

Conclusions:
In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells and is therefore considered to be non-mutagenic in this HPRT assay.
Executive summary:

A GLP-compliant mammalian cell mutagenicity test according to OECD guideline 476 was performed to investigate the potential of the test article to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of 3320 µg/mL applied in the pre-experiment was equal to approximately 10 mM. The concentration range of the main experiments was limited by phase separation. The test item was dissolved in THF. The tested concentrations ranged from 0.8 to 416 µg/ml.

Phase separation, visible to the unaided eye, was noted at 52.0 µg/mL and above in experiment I with and without metabolic activation. In experiment II phase separation occurred at the maximum concentration of 416.0 µg/mL in the presence of metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures solely occurred in the second experiment at the highest analysable concentration of 13.0 µg/mL without metabolic activation (24 hours treatment). Exceedingly severe cytotoxic effects precluded analysis of the data at the next higher concentration of 26.0 µg/mL. No relevant and reproducible increase in mutant colony numbers/106 cells was observed in the main experiments up to the maximum concentration. The induction factor did not reach or exceed the threshold of 3 times the mutation frequency of the solvent controls. In both experiments of this study (with and without S9 mix) the range of the solvent controls was from 5.5 up to 36.5 mutants per 106 cells; the range of the groups treated with the test item was from 1.2 up to 48.1 mutants per 106 cells. EMS (150 µg/mL) and DMBA (1.1 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies. Although the mutation frequency of the EMS control in both cultures of the first experiment remained well within the historical range of positive controls, the induction factor just reached or slightly exceeded the limit of 3.0. This fact is based on a relatively high mutation frequency of both solvent controls running close to the upper limit of the historical range of solvent controls. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, the test article is considered to be non-mutagenic in this HPRT assay.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial mutagenicity

A Salmonella typhimurium and Escherichia coli Reverse Mutation Assay (Ames Assay) according to GLP and OECD guideline 471 was performed in order to assess the potential of the test substance to induce reverse mutations in histidine (his- to his+) and tryptophan (tryp- to tryp+) genes. An initial range finding assay revealed no cytotoxicity of the highest concentration of 5 mg/plate both in the plate incorporation and the preincubation exposure methods. Therefore, concentrations ranging from 0.1 µg/plate to 5000 µg/plate were tested. The assay was conducted with S. typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli strain WP2 both in the presence and absence of an exogenous mammalian metabolic activation system. There was a statistically significant increase in the frequency of revertant colonies in positive control groups validating the functioning of the assay. Statistical analysis showed no significant differences for the frequency of revertants between negative control and test substance groups. A confirmatory assay was performed using the preincubation method of exposure in order to verify the results of the initial Reverse Mutation Assay. Under the test conditions and based on the results of this study, the test substance is not mutagenic in the Ames test.

Mammalian cell mutagenicity

A GLP-compliant mammalian cell mutagenicity test according to OECD guideline 476 was performed to investigate the potential of the test article to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The concentration range of the main experiments (0.8 - 416 µg/ml) was limited by phase separation. The test item was dissolved in THF. Phase separation, visible to the unaided eye, was noted at 52.0 µg/mL and above in experiment I with and without metabolic activation. In experiment II phase separation occurred at the maximum concentration of 416.0 µg/mL in the presence of metabolic activation. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures solely occurred in the second experiment at the highest analysable concentration of 13.0 µg/mL without metabolic activation (24 hours treatment). Exceedingly severe cytotoxic effects precluded analysis of the data at the next higher concentration of 26.0 µg/mL. No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation. Appropriate reference mutagens (EMS and DMBA), used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore the test substance is considered to be non-mutagenic in this HPRT assay.

Chromosomal aberration

A chromosome aberration study according to OECD guideline 473 and compliant with GLP was performed to assess the test article's potential to induce chromosomal aberrations in Chinese Hamster Ovary (CHO) cells in the presence or absence of (Aroclor induced rat liver S9) metabolic activation system. An initial dose range finding study showed the test substance to be cytotoxic (100%) at concentrations of 5.0, 1.0 and 0.1 mg/mL whereas concentrations of 0.01 mg/mL exhibited about 50% cytotoxicity. Therefore, the definitive assay was conducted with concentrations of 0.01, 0.001 and 0.0001 mg/mL of the test substance incubated with CHO cells for 3 h with or without metabolic activation. The cell viability in the definitive assay was similar to that observed in the initial range finding assay. There was no increase in the number of structural chromosomal aberrations or polyploidy at any of the concentrations tested, as compared to the negative control. This result was confirmed in a second experiment with longer exposure. The increase in chromosomal aberrations with exposure of the positive controls in both the activated (Cyclophosphamide) and the non-activated (Mitomycin C) assays verified the sensitivity of the test system. Under the test conditions employed, the test article did not induce chromosomal aberrations in cultured mammalian somatic cells mid is considered non-clastogenic.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for the purpose of classification under Regulation (EC) No.1272/2008. Based on the present data, classification for genotoxicity is not warranted under Regulation (EC) No.1272/2008.