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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(1997)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Reaction mass of 2,2'-iminodiethanol and 2-{[bis(2-hydroxyethyl)amino]methyl}-4-[2-(4-hydroxyphenyl)propan-2-yl]phenol and 2,2-bis(4-hydroxyphenyl)propane and 2-{[bis(2-hydroxyethyl)amino]methyl}-4-[2-(3-{[bis(2-hydroxyethyl)amino]methyl}-4-hydroxyphenyl)propan-2-yl]phenol
- Appearance: viscous, clear, yellowish liquid

Method

Target gene:
HPRT locus
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM containing Hank's salts supplemented with 10 % FBS, neomycin (5 µg/mL) and amphotericin B (1 %); for the selection of mutant cells the complete medium was supplemented with 11 µg/mL 6-thioguanine.
- 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:
S9-Mix from the liver of phenobarbital/beta-naphthoflavone induced male Wistar rats.
Test concentrations with justification for top dose:
Exp. I (4 hours exposure): without S9-mix 4.8, 9.5, 19.0, 38.0, 57.0, and 76.0 µg/mL; with S9-mix 9.5, 19.0, 38.0, 76.0, 114, and 152 µg/mL
Exp. II: 24 hours exposure, without S9-mix 9.5, 19.0, 38.0, 76.0, 114, and 152 µg/mL; 4 hours exposure, with S9-mix 19.0, 38.0, 76.0, 114, 152, and 228 µg/mL

The following concentrations were selected for reading: Exp. I without S9-mix 9.5, 19.0, 38.0, 57.0, and 76.0 µg/mL and with S9-mix 19.0, 38.0, 76.0, 114, and 152 µg/mL; Exp. II without and with S9-mix 19.0, 38.0, 76.0, 114, and 152 µg/mL.
Vehicle / solvent:
ethanol
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
without metabolic activation: ethylmethane sulphonate (1.2 mM); with metabolic activation: 7,12-dimethylbenz(a)anthracene (4.3 and 8.6 µM)
Details on test system and experimental conditions:
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. 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). The highest concentration in the pre-test (2440 µg/mL) was based on the solubility properties of the test item. The pre-test was performed in a concentration range from 19.1 and 2440 µg/mL to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.

The main test was performed in two independent experiments. 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.

Treatment Protocol without Metabolic Activation: Approximately 1.5 x 10exp6 (single culture) and 5 x 10exp2 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment. After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 µl/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps. In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation. The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below. Three or four days after treatment 1.5 x 10exp6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5 x 10exp5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DETERMINATION OF CYTOTOXICITY: Toxicity of the test item is indicated by a reduction of the 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 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 and statistical significance were considered together.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments with and without metabolic activation. The mutant frequency did not exceed the historical range of solvent controls in both parallel cultures. The induction factor remained below the threshold of three times the mutation frequency of the corresponding solvent control.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH was shifted to 7.89 in the pre-experiment which is quite close to the basic borderline. Therefore, the pH was measured again at the maximum concentration of the first experiment without metabolic activation. With a pH of 7.35 there was no relevant shift towards the basic range.
- Effects of osmolality: There was no relevant shift of the osmolarity of the medium even at the maximum concentration of the test item.
- Precipitation: Precipitation occurred at 1220 µg/mL and above after 4 and 24 hours treatment without metabolic activation and after 4 hours treatment with metabolic activation. In the main experiments no precipitation was noted at the end of treatment up to the maximum concentration with and without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The concentration range of the main experiments was limited by cytotoxicity. Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50 % occurred in the second experiment without metabolic activation and in both experiments with metabolic activation at 152 µg/mL. In the first experiment without metabolic activation cytotoxicity as described above was noted at 38.0 µg/mL and above. The recommended toxic range of approximately 10-20 % cloning efficiency I or relative cell density was covered with and without metabolic activation.
Remarks on result:
other: strain/cell type: Chinese hamster lung fibroblasts (V79)
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Executive summary:

A study according to OECD TG 476 was conducted in order to investigate the potential of the substance to induce gene mutations at the HPRT locus in mammalian cells. For that purpose, V79 cells were exposed to the test item in the first experiment for 4 hours with and without metabolic activation and in the second experiment for 4 hours with and for 24 hours without metabolic activation. The maximum concentration of the pre-experiment (2440 µg/mL) was based on the solubility properties of the test item. The concentration range of the main experiments was limited by cytotoxicity.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, 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 in mammalian cells.